The transition from hunting and gathering to farming involved a major cultural innovation that has spread rapidly over most of the globe in the last ten millennia. In sub-Saharan Africa, hunter–gatherers have begun to shift toward an agriculture-based lifestyle over the last 5,000 years. Only a few populations still base their mode of subsistence on hunting and gathering. The Pygmies are considered to be the largest group of mobile hunter–gatherers of Africa. They dwell in equatorial rainforests and are characterized by their short mean stature. However, little is known about the chronology of the demographic events—size changes, population splits, and gene flow—ultimately giving rise to contemporary Pygmy (Western and Eastern) groups and neighboring agricultural populations. We studied the branching history of Pygmy hunter–gatherers and agricultural populations from Africa and estimated separation times and gene flow between these populations. We resequenced 24 independent noncoding regions across the genome, corresponding to a total of ∼33 kb per individual, in 236 samples from seven Pygmy and five agricultural populations dispersed over the African continent. We used simulation-based inference to identify the historical model best fitting our data. The model identified included the early divergence of the ancestors of Pygmy hunter–gatherers and farming populations ∼60,000 years ago, followed by a split of the Pygmies' ancestors into the Western and Eastern Pygmy groups ∼20,000 years ago. Our findings increase knowledge of the history of the peopling of the African continent in a region lacking archaeological data. An appreciation of the demographic and adaptive history of African populations with different modes of subsistence should improve our understanding of the influence of human lifestyles on genome diversity. The central African belt represents a key region for understanding recent changes in human history and modes of subsistence because the largest group of hunter–gatherers of Africa, the Pygmies, still inhabits this region and coexists with neighboring agricultural populations. However, the understanding of the peopling history of equatorial Africa is hampered by the rapid disintegration of fossil remains in the rainforest's acidic soils. When archaeology fails, population genetics can reconstruct the history of populations from their present-day genetic variation. We generated a large resequencing dataset in different farming, Western Pygmy, and Eastern Pygmy populations dispersed over the African continent. By means of simulation-based inferences, we show that the ancestors of Pygmy hunter–gatherers and farming populations started to diverge ∼60,000 years ago. This indicates that the transition to agriculture—occurring in Africa ∼5,000 years ago—was not responsible for the separation of the ancestors of modern-day Pygmies and farmers. We also show that Western and Eastern Pygmy groups separated roughly 20,000 years ago from a common ancestral population. This finding suggests that the shared physical and cultural features of Pygmies were inherited from a common ancestor, rather than reflecting convergent adaptation to the rainforest. There is archaeological and genetic evidence to suggest that anatomically modern humans originated in a small, isolated population in Africa 150–200 thousand years ago (Kya). Worldwide population radiation then occurred 50–75 Kya The Pygmy populations occupy a vast territory extending west-to-east along the central African belt from the Congo Basin to Lake Victoria. They have a mostly forest-dwelling hunter-gathering lifestyle, specific cultural practices  to determine how these three population groups separated over time  and (ii) to estimate the time at which these population groups separated and the levels of subsequent gene flow between them. We generated a large multilocus resequencing dataset for five agricultural and seven Pygmy populations dispersed over the African continent. We then compared the ∼7.8 Mb of diploid sequences obtained with a large number of simulations exploring various demographic and branching scenarios, to identify the models best fitting the observed data. We then estimated, with the approximate Bayesian computation (ABC) method To establish the branching history of agricultural, Western and Eastern Pygmy hunter–gatherer populations from Africa, and to coestimate separation times and levels of gene flow between these groups of populations, we resequenced 24 independent non coding genomic regions of ∼1.3 kb each, corresponding to a total of ∼33 kb per individual, including 20 autosomal regions, and one mtDNA, one Y-linked and two X-linked regions . This reK = 2), Pygmy groups and AGR populations were separated into two different clusters  = 75.8%), further partitioned the group of farmers into those inhabiting the Central African belt and those located in South-East Africa. No other cluster was found for K values higher than 4 , and with the complete shifting of their cultural practices towards those of neighboring agricultural populations K = 4 separated Mozambicans from the other agricultural populations  may affect historical and demographic inferences ambicans , Text S1ambicans , and S7.D statistic was also significantly lower across autosomal regions in the AGR group  have been shown to influence the estimation of population separation times and levels of gene flow GR group , this paGR group  observedP = 0.04, see P<0.01). Population structure models could also theoretically fit the PYG data, in the presence of gene flow with AGR populations. However, the occurrence of population structure in PYG populations alone is unlikely because (i) our analyses considered a pruned population dataset excluding admixed populations ulations . AltogetA-WE model, involving an ancient divergence between the ancestors of the AGR and PYG groups, followed by a split of PYG ancestors into the WPYG and EPYG groups; (ii) the W-AE model, in which the most ancient split is that between the ancestors of the WPYG and AGR groups; (iii) the E-AW model, in which the most ancient divergence is that between the ancestors of the EPYG and AGR groups, and (iv) the AWE model, in which all populations diverged simultaneously , and was therefore identified as the most probable model given the data : the smaller ξ, the better the simulations fitted the observed data, and the greater the enrichment of the A-WE model in these simulations. This analysis thus clearly supports our conclusion that the A-WE model is the most probable, given the autosomal data.Twenty autosomal regions were simulated 1,000,000 times under the four possible IM models  with IM approach . Indepenthe data . We then “small” . We obseA-WE model. Several lines of evidence suggest that sex-biased gene flow, ancient maternal population structure and/or stronger genetic drift have contributed to the high levels of mtDNA differentiation observed today between the two Pygmy groups . Consequently, a more recent divergence between the two Pygmy groups (than between the PYG and AGR groups) is required, both to compensate for the stronger genetic drift among PYG populations and to generate the observed lower level of differentiation of autosomal regions between the two PYG groups. Taken together, our analyses, which explored a wide range of models and parameter values  . This anA-WE model, As previously mentioned, all analyses  were also performed with the “composite population dataset”, which includes the admixed/structured individuals/populations , and S7.ca. 60 Kya is consistent with our recent single-locus estimation based on the mtDNA diversity of African farmers and Western Pygmies ca. 50–80 Kya, based on several genetic markers The implications of our estimates are important for broader issues in African prehistory, although they must be interpreted carefully because of their large confidence intervals . The finca. 20 Kya is also consistent with a previous mtDNA study, dating the time of separation of these two Pygmy groups to at least 18 Kya Our evidence for a separation of the ancestors of Western and Eastern Pygmy groups Our multilocus resequencing analyses, supported by simulation-based inferences, increase our knowledge of the peopling history of the African continent by revealing that: (i) Western and Eastern Pygmies share a recent common ancestry, indicating that their shared specific traits, such as hunting and gathering in rainforest ecosystems and short stature, were acquired by shared ancestry rather than by convergence, and (ii) the agricultural revolution associated with Bantu expansions is not responsible for the population differentiation currently observed between farmers and Pygmy hunter-gatherers, suggesting that the ancestors of these two populations had a hunting and gathering lifestyle but possibly in different, specific ecological habitats . The distribution of lithic industries in the Middle Stone Age points to the existence of hunter-gathering groups in the open savanna environment of Central Africa The null model of selective neutrality provided by this study will also prove useful for the identification of genetic variants contributing to complex diseases and for the detection of genomic regions targeted by natural selection. In particular, a detailed study of the genome-wide footprints of local positive selection in African farmers and Pygmy hunter-gatherers, integrating the demographic model determined in this study, should facilitate robust identification of the population-specific adaptive responses of these two human groups to their different climatic, pathogenic and nutritional environments. These studies should help to decipher the potential genetic basis of the population-specific traits characterizing these ethnic groups, such as the short mean stature of the Pygmies. More generally, an appreciation of the demographic and adaptive history of these populations will improve our understanding of the influence of human lifestyles on genome diversity in terms of both health and disease.http://alfred.med.yale.edu/alfred/index.asp) under sample UID numbers SA000005F, SA000006G, SA001805O, and SA000487T, respectively. All sampled individuals were healthy donors from whom informed consent was obtained. This study was approved by the Institut Pasteur Institutional Review Board (n° RBM 2008.06).Sequence variation was surveyed in DNA samples from 12 sub-Saharan African populations. The panel included 118 samples from five agricultural populations , 71 samples from four Western Pygmy populations , and 47 samples from three Eastern Pygmy populations (Mbuti from the Democratic Republic of Congo [N = 24] and Twa from southern [N = 8] and northern [N = 15] Rwanda) . The BiaThe 24 independent regions sequenced here represent a total sequence length of 32.75 kb per individual (mean sequence length per region of 1.31 kb). We selected 20 non coding, independent autosomal regions  to decipK value , we ran 20 independent runs with different seeds and found likelihoods to be stable across runs. We focused on several aspects of our resequencing dataset, including classical diversity indices , neutrality statistics  and population differentiation statistics (pairwise FST). All these statistics, the observed site frequency spectra and those expected under a constant population size model, as well as the significance of FST values, were obtained with DnaSP v.4.10.9 S mutations segregating in populations i and j. Then i, i and j and i and j with a relative frequency in merged populations lower than f.We reconstructed haplotypes with PHASE v.2.1 Then μ) and effective population sizes (Ne) drawn from gamma distributions  to assess the statistical significance of observed neutrality statistics and their means and variances across autosomal regions and (ii) to determine which models and parameters best fitted our empirical data. Simulations were performed using coalescent theory, as implemented in SIMCOAL v.2.1.2 ibutions , as in pFor each independent genomic region, the statistical significance of the neutrality statistics in each population group was assessed by comparing observed values with 100,000 values obtained from simulations of a sample, the size of which corresponded to that of the tested population sample, under a neutral model of evolution, assuming a constant population size and no recombination . The statistical significance of means and variances of neutrality statistics across the 20 autosomal regions was assessed by simulating 100,000 sets of 20 independent regions under the same assumptions. Models were tested by simulating 100,000 and 250,000 datasets under each demographic and IM models respectively, with model parameters randomly drawn from prior distributions (see section below).ξΨ) between the simulated and observed summary statistics, S′ and S. These distances were measured by calculating the normalized metric D D was considered to be small when lower than ξ = 0.5. This flexible statistical framework, which is based on comparisons between simulations and observed data, makes it possible to test complex models with fluctuations in effective population size, population separation times and gene flow, without estimating the real likelihood of the data (ξ = 0), which would be unfeasible given the complexity of the data and the models. The tested demographic and IM models were all simulated with prior distributions of model parameters  under this demographic expansion were similar to the observed statistics for the AGR group (data not shown). For Pygmy populations, we compared the empirical summary statistics obtained for the WPYG and EPYG population groups . We did not aim to identify a best-fitting model for the demographic regime of AGR populations, because historical n groups  with sumn groups . We consn groups  presentin groups . The num dataset  and S6.FST , considering the previously described population-specific demographic models for each population group : a modelise FST . We test dataset  and S7.A-WE model; A-WE model, using large flat prior distributions for separation time and migration rate parameters, except that the time of the oldest divergence was necessarily constrained by the time of the latest divergence <ξ criterion Click here for additional data file.Figure S2Ψ0.5) obtained in comparisons with simulated statistics, based on the composite population dataset. Times are in generations. Tbot and Sbot are the time and strength of the bottleneck, respectively. Trec and Srec are the time and strength of the population size recovery, respectively. Modeling details and the prior distributions of parameters are given in Ψ0.5 for a given model and set of parameters, by resampling, among 100,000 simulations, 100 sets of 10,000 simulations of the model, calculating Ψ0.5 for each set and reporting the mean Ψ0.5 across sets. The model with one bottleneck  and one recovery  generated, for WPYG, the maximum Ψ0.5 in 62% of cases when compared with all models and in 98% of cases when compared with only constant population size models. For the EPYG group, the constant population size model generated the maximum Ψ0.5 in 56% of cases when compared with all models.Different models simulating the demographic regime of the WPYG and EPYG groups and the mean proportion of small distances ((10.20 MB TIF)Click here for additional data file.Figure S3A-WE model for the composite population dataset. Divergence times Tdiv are expressed in years and migration rates m in proportion of migrants per generation. Black lines represent prior distributions and gray histograms represent approximated posterior distributions obtained by the ABC method ξ - the threshold at which distances between simulated and observed statistics are considered to be “small” . The joint approximated posterior distribution of TdivWPYG-EPYG and mWPYG-EPYG is shown in Prior and approximated posterior distributions of the IM model and IM parameters under the best-fit  “small”  - and th(6.07 MB TIF)Click here for additional data file.Figure S4Ψ0.5. The time of divergence TdivWPYG-EPYG and the migration rate mWPYG-EPYG are reported in generations and in proportion of migrants per generation, respectively.Approximated joint posterior distribution of the time of divergence and migration rate between Western and Eastern Pygmies for the composite population dataset. The posterior distribution of the two parameters is estimated by means of the proportion of small distances (4.52 MB TIF)Click here for additional data file.Figure S5Tdiv and m, respectively.Behavior of selected summary statistics under various levels of divergence and gene flow. Time of divergence (in generations) and migration rate (in proportion of migrants per generation) are represented by (4.93 MB TIF)Click here for additional data file.Table S1Location of the 25 resequenced regions and their respective distances to coding regions.(0.09 MB DOC)Click here for additional data file.Table S2FST values among the 12 sub-Saharan African populations for (A) 20 autosomal regions, (B) two X regions, (C) one Y region and (D) one mtDNA region.Mean pairwise (0.14 MB DOC)Click here for additional data file.Table S3Mean diversity indices and neutrality tests across the 24 independent genomic regions sequenced in the composite population dataset of WPYG, EPYG and AGR.(0.05 MB DOC)Click here for additional data file.Table S4Variances of statistics from sequence-based neutrality tests across the 20 autosomal regions in WPYG, EPYG and AGR populations, using the filtered and composite population datasets.(0.03 MB DOC)Click here for additional data file.Table S5Mean summary statistics for genetic differentiation between the WPYG, EPYG and AGR populations, across the 24 genomic regions, for the filtered and composite population datasets.(0.06 MB DOC)Click here for additional data file.Table S6Prior distributions of the parameters of 33 models simulated to assess the demographic regime of Pygmy population groups, using the composite population dataset.(0.12 MB DOC)Click here for additional data file.Table S7Prior distributions of the parameters of the IM models simulated to assess the branching history of the AGR, WPYG and EPYG populations, using the composite population dataset.(0.04 MB DOC)Click here for additional data file.Table S8Prior distributions of the parameters of 33 models simulated to assess the demographic regime of Pygmy population groups, using the filtered population dataset.(0.11 MB DOC)Click here for additional data file.Table S9Prior distributions of the parameters of the IM models simulated to assess the branching history of the AGR, WPYG and EPYG populations, using the filtered population dataset.(0.04 MB DOC)Click here for additional data file.Table S10Prior distributions and means of mutation rates and effective population sizes used for all coalescent simulations.(0.03 MB DOC)Click here for additional data file.Text S1Rationale of the study and supplementary analyses.(0.08 MB DOC)Click here for additional data file.


The study of inbreeding depression has major relevance for many disciplines, including conservation genetics and evolutionary biology. Still, the molecular genetic basis of this phenomenon remains poorly characterised, as knowledge on the mechanistic causes of inbreeding depression and the molecular properties of genes that give rise to or modulate its deleterious effects is lacking. These questions warrant the detailed study of genetic loci giving rise to inbreeding depression. However, the complex and polygenic nature of general inbreeding depression makes this a daunting task. Study of inbreeding effects in specific traits, such as age-specific mortality and life span, provide a good starting point, as a limited set of genes is expected to be involved.Drosophila melanogaster. The inbreeding effect was expressed at moderately high temperature, and manifested itself as severe premature mortality in males, but not in females. We used a North Carolina crossing design 3 to estimate average dominance ratio and heritability. We found the genetic basis of the lethal effect to be relatively simple, being due mainly to a single recessive QTL on the left arm of chromosome 2. This locus colocalised with a QTL that conditioned variation in female life span, acting as an overdominant locus for this trait. Male life span was additionally affected by variation at the X-chromosome.Here we report on a QTL mapping study on inbreeding related and temperature sensitive lethality in male This demonstrates that analysis of large conditional lethal effects is a viable strategy for delineating genes which are sensitive to inbreeding depression. Inbreeding depression is defined as the decrease in fitness related characters in offspring resulting from matings between related individuals. This phenomenon is known to have large impact on fitness, adaptive ability and extinction probability of populations, and has an important role as a selective force in the evolution of mating systems and dispersal strategies -3. FurthAlthough inbreeding depression is well studied at the phenotypic and population level, knowledge on the mechanistic genetic basis of inbreeding depression is still very scant. Only recently have powerful tools become available to detect loci that underlie inbreeding depression  and to cInbreeding depression is known to occur in virtually every fitness component, but life span is a particularly interesting character . Life spDrosophila melanogaster displaying temperature sensitive age-specific adult lethality. Conditional adult lethal effects are encountered regularly in inbred populations of Drosophila [2 males were backcrossed to females from both parental lines . The heritability in the narrow sense was found to be 0.31. Females from the control line were found to display some premature female mortality  days for males and 40.0 ± 0.09 (s.e.) days for females. The control crosses clearly showed conspicuous expression of the male lethal effect between day 5 and 12, whereby the lethal behaved as a recessive Figure . This pey Figure . This clWe found multiple QTL conditioning life span and age-specific mortality on all chromosomes. As we are lacking the resolution to perform fine-mapping of all loci, we adopted a conservative approach to detect QTL. We listed all QTL that were detected both by the single marker analysis and the composite interval mapping, and which had peaks separated by at least one marker interval Table . One of 1 crosses were significantly different, both for males and females. Reciprocal crosses of females were not different , dismissing maternal effects as an important factor. However, males from the reciprocal crosses did show a significant difference , suggesting some X-linked QTL. The X-chromosome of the LI4 line conferred superior longevity (+2.4 days). Our design cannot further delineate these X-linked QTL affecting male life span, as F2 sires do not transmit the X-chromosome to their backcross sons.We tested whether the reciprocal FThe estimate of average dominance ratio in female life span suggested one or several overdominant loci. Indeed, we detected a minimum of six major QTL segregating for female life span, and all but one of these behaved as overdominant loci Table . Note ho2 backcross to the LI4 lethal line , suggesting a shared genetic basis. However, no such correlation was observed within the B1 backcross to the control line . We wanted to determine whether the lethal allele was responsible for genetic variation in female life span too, even though female flies do not show a lethal phase  and one of the QTL affecting female life span hs, a heat-sensitive allele previously found segregating in our G83 base stock [chico, DNA methyltransferase 2, Sir2, tamas, Target of rapamycin, bubblegum, Myosin 31DF, porin and Pten, whereas Hsp60D is involved in the response to heat. It has been demonstrated that genes with large mutational effects on longevity are not necessarily segregating variation in natural populations [The ultimate aim of this study is to identify the main QTL at the molecular level. The position of our lethal does not correspond to that of se stock ,24. Howeulations . Therefoulations ,35. HoweIt is not our intention to generalise the findings of this single instance of inbreeding depression to the general genetic basis of inbreeding depression. However, by focusing on well characterised effects in a defined trait, instead of general inbreeding depression in fitness, we have shown that it is possible to identify major genetic factors and these will likely be amenable to detailed molecular characterisation. This type of data can enable powerful studies that can provide complementary insights, e.g. whether inbreeding in life span will map to candidate genes known to affect longevity. In addition, since stress responsive mechanisms are known to influence life span , it willThe inbred line I4 was chosen because it displays a high level of early onset adult mortality. Discovery of this lethal effect and the origin of the line are described elsewhere . In brieLines with high levels of homozygosity are desirable in QTL-mapping designs, as this maximizes linkage disequilibrium between QTL and markers. For this purpose, we established highly inbred lines from I4 and I13 by performing 7 additional generations of inbreeding (expected F ~0.95). These lines will be referred to as LI4  and CI13 (Control Inbred), as to distinguish them from their progenitor stocks.Lines LI4 and CI13 have been maintained in large numbers  in quarter-pint bottles  at 25°C and 40–60% relative humidity. For assay of adult survival we supplied the food medium with ampicillin (100 mg/L) to avoid bacterial growth.http://i122server.vu-wien.ac.at/Microsatellite%20Loci/Loci%20Titelpage.html, Charles Aquadro laboratory and David Goldstein microsatellite page). The URLs of the latter two websites are no longer available, but information on some of these markers has been published [For the contrast LI4 vs. CI13 we used microsatellite markers to track segregation of the parental genomes  and LI4 (P2) flies. We set up several single-pair vials at 25°C for each reciprocal cross (P1 × P2 and P2 × P1), discarded the parents and allowed F1 flies to hatch. Thereafter, ten bottles containing 5 F1 females and 5 F1 males from each reciprocal cross  were set up. Simultaneously, culture bottles were set up for each parental line using normal culturing procedure. This set consisted of 2 subsets, offset by two days to allow for transfer of males to "fresh" females in the backcrosses (as described below). To initiate the backcrosses, we collected F2 males as well as virgin females from each of the respective parental lines. All were kept separate in yeast-supplied vials for 3 days at 20°C. Thereafter, F2 males were individually placed in vials containing either three P1 or P2 virgin females. In addition, we set up control families with males collected from the parental stocks. We set up equal amounts of B1- and B2-crosses. After 2 days males were aspirated into the alternative cross-vial with fresh females. The food-tablets with eggs were transferred to 68 ml plastic bottles with 21 ml of standard medium, in order to avoid crowding effects. Two days later, females were discarded and males were removed and stored at -20°C. As in the first set, food tablets were transferred to plastic bottles. Backcross progeny of 240 families was collected.Traditionally, inbreeding depression is analysed by contrasting inbred lines with the ancestral outbred population. We chose not to pursue this strategy, as we aimed to detect the genetic basis of a specific lethal effect, rather than general inbreeding. Instead, we used an inbred control line, which maximizes the power of the mapping design. We decided to employ a half-sib mating test design based on a North Carolina Design 3 (NCD3) . In thisa Figure . The advFor every family, 20 males and 20 females were collected as virgins and stored in single-sex vials containing ten individuals . These were placed at 29°C, refreshed at day 2 and thereafter twice per week until all flies were dead. Dead flies were scored three times per week. Individual life span was taken as the midpoint of the scoring interval in which the fly died. We lost 0.4% of the experimental flies due to escapes and accidental deaths.Analysis of variance of the North Carolina Design 3 cross was performed on the life span data as described by Kearsey and Pooni  followinyijl is the life span of offspring i of sire j tested against tester l, m is the intercept, Tl is the effect of tester l, sj the effect of sire j, (Ts)jl the interaction of sire j against tester l and eijl is the random error associated with offspring i with sire j tested against tester l. Partitioning of mean squares (MS) allows estimation of additive variance, dominance variance and environmental variance . Assuming no epistasis or gametic phase disequilibrium is present, average dominance ratio (DR) is given by:where VD being the dominance variance and VA the additive variance  [h2) is given by:with uencies) . The herVE being the environmental variance [with variance . All staμx), as estimated byIn addition to mean life span, we analysed weekly age-specific mortality . The program provides QTL peaks and 2-LOD support intervals, which have a high probability (~95%) of containing the QTL [X2 2 = 6.23, P = 0.045 and BIB; X2 2 = 7.20, P = 0.027). We estimated additive (ak) and dominance (dk) effects for QTL k, k = 1,..., n by pooling information from both backcrosses and fitting an additive linear model:where ographer ,46, usin the QTL . Signifi the QTL . Map disyil is the observation for family i in the cross to parental line l. Furthermore, m is the intercept, Tl is the effect of crossing to tester line l, xai and xdi are the explanatory variables of ak and dk which depend on flanking marker genotype and backcross type, and eil is the residual term [where ual term ,49.1 crosses of the control families were significantly different using a t-test.X-linked and carry-over effects will affect these estimates, so we tested whether the reciprocal FCJV designed and performed the experiment, analysed the results and wrote the manuscript. RB made, and performed initial characterization of, the I4 and I13 progenitor lines, provided guidance throughout the study and helped writing the manuscript. VL participated in the design of the study, supervised the experiment and helped writing the manuscript. All authors read and approved the final manuscript.Microsatellite list. Contains microsatellite names, accession numbers, cytological locations, genetic locations, repeat type, references and allelic size ranges.Click here for file


P < .05) than in the 16 g needle aspiration and in the scraped ovaries (57% versus 36% and 47%) while the rate of cumulus-intact oocytes was higher than aspiration (46.9% versus 39.36%) but lower than scraping (46.97%) (P < .001). In unpeeled ovaries there was significant difference in maturation rate of oocytes recovered by Tuohy needle in respect to peeled ovaries . Combination of aspiration and scraping by Tuohy needle allows a faster and reliable collection of oocytes suitable for horse IVM.Two methods have been described to recover oocytes from equine follicles in excised ovaries: aspiration and scraping. Aim of this work was to develop an effective method for collecting equine oocytes using Tuohy needle and comparing this technique to aspiration and scraping, with or without tunica albuginea removal. This hollow hypodermic needle, usually employed for inserting epidural catheters, is designed with a slightly curved tip, shaped similar to a small curette. In unpeeled ovaries, the recovery rates of Tuohy needle group was higher ( Two methods have been described to recover oocytes from equine follicles in excised ovaries: aspiration and scraping. Aspiration has been found to give a low recovery rate (31–46%) [in vitro fertilization to ICSI, embryo culture, and cloning.Progress in ry rate 3–46% [1, (31–46%) , and to (31–46%) , 3. The (31–46%) , 6. It iAim of this work was to develop an effective method for collecting equine oocytes assembling the feature of aspiration (fastness) with that of scraping (high recovery rate of cumulus-intact oocytes). For this purpose a particular hypodermic needle, usually employed for inserting epidural catheters, known as Tuohy needle, was used. This hollow needle is designed with a slightly curved tip, shaped similar to a small curette. Furthermore, we compared cumulus morphology and maturation rates to metaphase II, among oocytes collected by this new technique and oocytes harvested by standard needle aspiration or scraping, with or without tunica albuginea removal from the ovary.Ovaries were collected at a local abattoir, freed of any surrounding extra tissue and brought to the laboratory soon after slaughter in PBS supplemented with penicillin/streptomycin at 30°C. For all the experiments, part of the ovaries were used without removal of the tunica albuginea (unpeeling) or after peeling of the tunica to expose follicles. After voiding of all visible follicles, peeled or unpeeled ovaries were bisected along their midline at 10 mm intervals, from ovulation fossa to the greater curvature, and any newly exposed follicles were utilized.All experiments were replicated three times and data were analyzed by one-way Analysis of Variance (ANOVA) using Student-Newman-Keuls Multiple Comparisons Test.In the first experiment all follicles were opened with a scalpel blade and the granulosa cell layer was scraped with a curette following the method of Hinrichs et al. . GranuloIn the second experiment all follicles were aspirated using a 16-g (1.6 × 40 mm) needle  attached via silicone tubing to a Kmar 3000 vacuum pump  in turn connected to an embryo filter  maintained at 38°. The aspiration pressure was set at 70 mm Hg. During aspiration the needle was repeatedly rinsed with HEPES-buffered TCM medium. After collection the excess fluid was drained through the filter and the sediment poured into square petri dishes. In the third experiment all follicles were aspirated using a 16-g Tuohy needle (1.6 × 80 mm)  Figures  in the sIn all experiments, collected cumulus-oocyte complexes (COCs) were classified as having compact, expanded (Ex), corona radiata only, or partial cumulus investments, depending on the presence and expansion of cumulus cells. Only Ex oocytes regardless of cytoplasmic appearance were used for this study, while all other COCs were used for other experiments.The selected Ex oocytes collected throughout the experiments were matured in vitro (IVM) following the procedure by Dell'Aquila et al. . After IMean number of follicles/ovary and oocytes/ovary, recovery rate and the cumulus morphology of oocytes collected in all experiments are reported, respectively, in Tables In the first experiment, 969 follicles were scraped with the curette from a total of 160 unpeeled ovaries with an average of 6.04 follicles per ovary. 973 follicles were scraped from a total of 143 peeled ovaries with an average of 6.80 follicles per ovary. To collect 100 oocytes, 4 people working for 4-5 hours with peeled ovaries and 3-4 hours with unpeeled ovaries were required.In the second experiment 256 follicles were aspirated with the 16-g needle from a total of 41 unpeeled ovaries with an average of 6.24 follicles per ovary. 312 follicles were aspirated from a total of 38 peeled ovaries with an average of 8.21 follicles per ovary. To collect 100 oocytes, 3 people working for 2-3 hours with peeled ovaries and 1.5–2 hours with unpeeled ovaries were required.P < .05) than in the 16-g needle aspiration groups and in the unpeeled scraped ovaries (P < .001)  . There were no differences in maturation rates of oocytes recovered in nonpeeled ovaries by Tuohy needle in respect to scraping in peeled ovaries.Both collection techniques, classical aspiration, or scraping are widely employed on excised ovaries and the comparison between these two methods have been reported previously , 3.In this study, we found that aspiration of follicles resulted in a lower oocyte recovery rate and higher prevalence of oocytes having partial cumulus than did follicle scraping which, in general, allow to recover oocytes with better quality. Similar results have been reported previously , 2, but In our study, the recovery rates obtained using the combination of aspiration and scraping by means of the Tuohy needle still remained significantly lower than that attained in peeled ovaries by scraping but maturation rates were not statistically different in these two groups. It is noteworthy that our technique improved the recovery rate of intact cumulus oocytes and maturation rate compared to aspiration by the conventional 16-g needle.Moreover, the maturation rates of expanded cumulus oocytes collected by Tuohy needle in unpeeled ovaries and by scraping in peeled ovaries are similar. In conclusion, the collection technique used in this experiment assembling aspiration and scraping allow to recover intact-cumulus oocytes without altering the maturation rate, so this finding is very important because both peeling and scraping are time consuming and limiting factors for in vitro oocytes maturation in the horse.


The production of sex steroids by follicular cells is proposed to be influenced by the maturity of the incumbent oocyte. Thus steroid levels may reflect suitability of an oocyte for IVF. We examined follicular fluids and granulosa cell production of steroid from IVF patients in order to test the relationship between steroid levels and fertilization.Follicular fluid and granulosa cells were extracted from 206 follicles of 35 women undergoing controlled ovarian stimulation. Follicular fluid was assayed for estradiol, progesterone and testosterone. Granulosa cells were cultured from individual follicles and their culture media assayed for production of these hormones after 24 hrs in vitro. Levels of steroids were correlated with follicular diameter, oocyte recovery and subsequent fertilization.Follicular fluid levels of progesterone were 6100 times higher than that of estradiol, and 16,900 times higher that of testosterone. Despite the size of follicle triggered after controlled luteinisation, the levels of progesterone and testosterone were maintained at relatively constant levels . However, estradiol levels were slightly lower in the larger follicles . With respect to oocyte recovery, no steroid showed a significant association in follicular fluid levels. Similarly no difference in follicular fluid steroid levels was found for those oocytes that did or did not fertilize. Significant quantities of progesterone were produced by the granulosa cells but production was constant regardless of the size of follicle from which the cells originated. Estradiol levels were only detectable in 10 of 121 cultures examined, and testosterone in none. Interestingly, when an oocyte was present follicular estradiol levels correlated with progesterone levels. However, when absent, follicular estradiol levels correlated with testosterone levels but not with progesterone.The principle steroid product of luteinized pre-ovulatory granulosa is progesterone, a differentiation triggered by the gonadotropin surge. However, absolute steroid levels are associated with follicular size, not oocyte maturation/ability to fertilize. Oocyte maturation is a complex process that includes the re-initiation and completion of the first meiotic division and subsequent progression to metaphase II, as well as nurse cell preparation of the oocyte cytoplasm. As the whole process of nuclear and cytoplasmic change is crucial for fertilization and early embryo development, investigations have concentrated on the composition of the intercellular environment of this process, the follicular fluid.Estradiol, progesterone and testosterone are the main steroid hormones that play essential roles during the follicular and luteal phases of the menstrual cycle. However other cytokines, such as inhibins, activins, insulin growth factor-2, insulin growth factor binding proteins, tumour growth factor-β and endothelial growth factor, have been measured in follicular fluid and correlated with oocyte maturation -4. NeverDespite this, there are still inconsistencies in the literature regarding estradiol and the other steroid hormone concentrations in follicular fluids, and also in granulosa cell cultures. There is even less information on steroid secretion profiles of granulosa cells cultured from individual follicle. Ryan and Smith carried out the first study of the biosynthesis capability of the human ovarian follicle in 1961 . They shThis study set out to investigate the levels of estradiol, progesterone and testosterone found in individual follicular fluid from stimulated cycles and their granulosa cell cultures after oocyte retrieval. The principal aim was to investigate steroid hormone levels in relationship to follicular diameter, oocyte presence and subsequent fertilization. However, this also allowed us to investigate steroidogenesis within the ovarian-follicular environment following stimulation for IVF, a situation akin to a follicle after the gonadotropin surge.In Vitro Fertilization/Intracytoplasmic Sperm Injection at the Fertility Unit of St Bartholomew's Hospital from 1999 to 2001 were recruited into this study. The ages of patients varied from 29 to 38 years old, and the follicular size varied from 10.2 mm to 35 mm. The total number of the follicles sampled was 206; 4 to 6 follicles per patient. The causes of infertility in this patient cohort were male, tubal or unexplained factors. Patients with endometriosis and polycystic ovarian syndrome were excluded from this study, as ovarian steroid hormone production is known to be altered in these conditions. Due to blood contamination, limited sample volume and lack of follow-up information, the final distribution of sample numbers between oocyte recovery, fertilization outcome and steroid analysis is shown in figure 35 patients who undertook Written consent was obtained from each patient, and this study was approved by the East London and The City Health Authority Research Ethics Committee (study number: P/98/222).g for 10 minutes to precipitate harvested granulosa cells. Follicular fluid supernatant was aspirated, divided into aliquots, and frozen at -20°C for subsequent hormone analysis. The fertilization outcome was also recorded according to the appearance of two pro-nuclei after incubating in cultured medium after 48 hours.Sample collection was 36 hours after subcutaneous administration of human chronic gonadotropin , and when at least three follicles in each patient had reached 18 mm in diameter as measured by ultrasound scan . Individual follicles were randomly selected prior to aspiration and measured in two dimensions by transvaginal ultrasound in order to obtain a mean diameter. The same operator performed all measurements and aspirations. Following identification of a suitable follicle, the follicle was gently pierced using a double lumen needle and aspirated, allowing the follicle to collapse slowly around the needle. The follicle was then flushed with 4.5 ml of heparinised saline (3×1.5 ml automated flushes) and aspirated again. Heavily blood stained aspirates were discarded and a further follicle was measured and aspirated if appropriate. A detailed record was kept of the volume of fluid aspirated per individual follicle prior to washing. Following examination of the follicular fluid, it was placed in a sterile tube and the presence of an oocyte was noted and recorded. If an oocyte was not retrieved, the follicle was flushed again with an additional 3 ml heparinised saline. If an oocyte was still not obtained, the follicle was recorded as having no oocyte retrieved. Then the follicular fluids were taken immediately to the Williamson Laboratory. Each tube was centrifuged at 200×3  supplemented with 10%v/v fetal calf serum (FCS)  and 1%v/v of an antibiotic stock solution containing 10,000 μg/ml Penicillin G sodium and 25 μg/ml Streptomycin . After washing and re-centrifugation at 200×g for 10 minutes, cells were resuspended in 5 ml of medium containing 0.2% hyaluronidase  for cell dispersion, and then incubated at 37°C for 30 minutes. After this time, the sample was again centrifuged at 200×g for 10 minutes and the cell pellet resuspended with 2 ml of medium and layered over 4 ml of 50% Percoll . This was centrifuged at 250×g for 20 minutes to separate the luteinized granulosa cell buffy coat from any contaminating erythrocytes. The granulosa cells were removed using a pipette and washed with 8 ml of medium. After a final centrifugation at 250×g for 10 minutes the pellet was resuspended and the cell number was determined using a haemocytometer. Cells were then plated, in triplicate wells, at a concentration of 10,000 cells per well in 24 well culture plates  and incubated at 37°C in a 95% air, 5% CO2 humidified environment for 24 hours and the cell free supernatant media was then collected and stored at -20°C for later analysis.Harvested follicular fluid cell pellets were resuspended in 4 ml of medium. This consisted of RPMI-1640 with glutamine and NaHCOSteroid hormone levels of progesterone, estradiol and testosterone were measured in follicular fluid and culture media by radioimmunoassay (RIA). These assays were carried out by commercial kits provided by Skybio Limited .Testosterone - The minimum detection of this essay was 0.2 nmols/L, and its standard range was from 0.7 nmols/L to 69 nmols/L. The cross reaction of this assay is reported to be lower than 0.01% for androstenedione, dihydroxytestosterone, estrone, estradiol-17β and progesterone. Quality control data from repeat assays showed < 10% CV.Estradiol - The minimal detectable concentration was 25 pmols/L, and its standard range was from 37 pmols/L to 11,100 pmols/L. The cross reactivity of this assay is reported to be: estrone < 6.2%, estradiol < 1.5%, cholesterol, DHEA, 17α-Hydroxyprogesterone, pregnenolone, progesterone and testosterone all lower than 0.01%. Quality control data from repeat assays showed < 15% CV.Progesterone- The minimum detectable concentration was 4 nmols/L, and its standard range was from 5 nmols/L to 80 nmols/L. Consequently samples needed to be diluted between 10 and 100 fold. The cross reactivity of this assay is reported to be: 17α-Hydroxyprogesterone < 0.5%, pregnenolone < 0.3%, testosterone, estradiol-17β, cholesterol all < 0.001%. Quality control data from repeat assays showed < 8% CV.Data was tabulated in Excel and imported in to a data analysis software StatsDirect package  and a p < 0.05 was taken as statistical significance. Graphical analysis was initially conducted as scatter plots where the concentrations and calculated total content of the steroid hormones were plotted against its individual follicular diameter and correlations tested. Distribution analysis revealed that the data generated had a non-Gaussian distribution; consequently, non-parametric statistical description and statistics have been used. Spearman rank correlation analysis was used to compare the inter-relationship between the examined steroids.For all follicular fluid samples, estradiol concentrations varied from 2.2 - 130.0 nmoles/L (median = 16.1 nmoles/L). Levels significantly decreased as follicular size increased (figure Testosterone concentration varied from 0.3-110.0 nmoles/L (median = 5.8 nmoles/L). The general trend was for testosterone concentration to remain constant despite follicular size figure . ConsistAll three steroid hormone levels were measured at 24 hours in culture media taken from granulosa cells isolated from individual follicles, and grown in triplicate. Testosterone was not detected in any of the 110 granulosa cell cultures examined. Only 10 out of 121 granulosa cell cultures examined contained detectable levels of estradiol production . Significant levels of progesterone production were detected in all 104 granulosa cell cultures examined . Progesterone production by granulosa cells was relatively constant and did not correlate with the diameter of the follicle from which they originated (data not shown).th centiles of the study populations), it became clear that fertilization rates increased dramatically for follicles of greater than 13 mm . However, the fertilisation rates slowly declined such that the very large follicles (> 25 mm) had a recovered oocyte fertilization rate similar to that of small follicles (< = 13 mm)  to C19 steroids (androgens) ,19. ImmuThe theca is a very thin layer of cells limited to, and defining, the peripheral boundary of the follicle. These cells express CYP17 as well as 3β-HSD such that further metabolism of either pregnenolone or progesterone (via 17-OH-pregnenolone and 17-OH-pregesterone) to androgens occurs. Thereafter, androgens can diffuse across the basal lamina into granulosa cells where they can be metabolized to estrogens, since these cells specifically express CYP19/aromatase  see fig.in vitro studies of cultured granulosa cells in rats also showed the increased levels of mRNA, protein and emzymatic activity of 3β-HSD after treatment with FSH [et al have shown that 3β-HSD was detected on both theca and granulosa cells from pre-ovulatory follicles [In our study, granulosa cell cultured samples were collected after 36 hours of hCG treatment and pre-oocyte retrieval, i.e. the cells were luteinized but pre-ovulatory granulosa cells. Steroidogenic ability changes under the influence of gonadotropin surge. Two previous with FSH ,23. Pellollicles . Here wePost-luteinisation (which is synonymous with ovulation), the LH/FSH surge induces or up-regulates CYP11A and 3β-HSD expression by granulosa cells such that this is now the dominant pathway and source of progesterone.Furthermore, the important enzyme catalyzing the crucial step for the formation of either DHEA or progesterone has been shown to have two types of isoenzymes, type 1 and type 2 . The speThe interrelationships among the three steroids may also reveal the maturation of granulosa tissue after the gonadotropin surge table . For exaet al [et al [The relationship between follicular size and oocyte maturation has been debated for some time. Eppig et al  claimed et al -30. Furtl [et al  demonstrThe fertilization and 2/4 cell blastocyst formation rate is lower for oocytes recovered from follicles of less than < 10 mm -34. IndeUnfortunately, most of these studies showing a significant correlation have grouped follicles into mature and immature (sometimes intermediate) by criteria based on an arbitrary follicle size cut-off. This will bias results: oocyte maturity is a function of follicular size, whilst steroid production by the follicle is also a function of follicular mass. If follicles are split simply into large (mature) or small (immature), it is inevitable that higher levels of steroids will be found in those follicles from which there was a higher rate of fertilization. Any relationship between fertilization outcome and prevailing steroid levels will be a causal function of follicle size.In this study, we examined the molar concentrations and total content of all three steroids in individual follicles. Although most of the steroids are bound to steroid-binding proteins, they are freely released in immunoassays; as such binding has much low affinity than that of an antibody which at equilibrium obliterates the steroid protein-binding effects . Therefode novo synthesised steroid of luteinised granulosa; when an oocyte is present follicular estradiol correlates with progesterone levels. However, if an oocyte is not present follicular estradiol levels correlate with testosterone and not progesterone levels.For stimulated cycles, luteinized pre-ovulatory follicular fluid levels of testosterone, progesterone and estradiol do not correlate with oocyte maturity (recovery and fertilization rate). Although oocyte fertilization rates correlate with follicular size up to ~21 mm, after this the rate declines, suggesting oocyte post-maturity. In post-luteinized follicles, the steroidogenic ability of granulosa cells is altered to become the dominant cell type producing progesterone. However, these studies do suggest that the oocyte has an influence on this metabolic switching: Although progesterone is the major The authors declare that they have no competing interests.XSW and DL carried out the research and collected the data. AJT and SMD proposed the study and collected the samples. XSW and RKI performed data analysis, interpreted data. XSW drafted the manuscript, RKI and SMD made significant editing for the manuscript. All authors have read and approved the final manuscript.


Intranasal medications are used to treat various nasal disorders. However, their effects on olfaction remain unknown. Zicam , a homeopathic substance marketed to alleviate cold symptoms, has been implicated in olfactory dysfunction. Here, we investigated Zicam and several common intranasal agents for their effects on olfactory function. Zicam was the only substance that showed significant cytotoxicity in both mouse and human nasal tissue. Specifically, Zicam-treated mice had disrupted sensitivity of olfactory sensory neurons to odorant stimulation and were unable to detect novel odorants in behavioral testing. These findings were long-term as no recovery of function was observed after two months. Finally, human nasal explants treated with Zicam displayed significantly elevated extracellular lactate dehydrogenase levels compared to saline-treated controls, suggesting severe necrosis that was confirmed on histology. Our results demonstrate that Zicam use could irreversibly damage mouse and human nasal tissue and may lead to significant smell dysfunction. As one of the five senses, the ability to smell plays a crucial role in defining the quality of life. Indeed, loss of sense of smell or anosmia can have detrimental consequences as our ability to detect and process noxious chemosensory stimuli is impaired One possible cause for the development of smell dysfunction is the use of various intranasal medications. Numerous intranasal drugs are available to treat various nasal disorders, including sinusitis, allergic rhinitis and nasal congestion. Although the safety and efficacy of the majority of these agents are well known, their effects on olfaction are not established. Moreover, some intranasal agents classified as “homeopathic” are marketed to treat common nasal disorders such as symptoms associated with the common cold. These agents are gaining popularity with consumers despite the lack of scientific data on their safety and efficacy In this study, we examined the short-term and long-term effects of several commonly used intranasal agents using mouse and organotypic cultures from human nasal tissue. Specifically, we tested saline, Afrin , Nasacort , lidocaine  and epinephrine , which are frequently utilized by physicians to treat various nasal disorders. Additionally, we tested one of the “homeopathic”, zinc-based intranasal agents, Zicam  as it has been previously implicated in smell dysfunction in vitro was observed in all animal groups except for those treated with Zicam (S2).To determine the effects of various intranasal agents on the functional properties of olfactory sensory neurons (OSN), we performed electro-olfactogram (EOG) analysis on mouse main olfactory epithelium (MOE) 3 and 9 days after intranasal administration of either saline, Afrin, Nasacort, epinephrine, lidocaine or Zicam. Following odorant stimulation, the activation of OSN We also examined the MOE for the expression of several proteins expressed in OSN. In all MOE examined, except those treated with Zicam, immunofluorescence staining demonstrated robust expression of AC3 (adenylyl cyclase 3) in the olfactory cilia, neural-specific β-tubulin and OMP (olfactory marker protein) in the cell bodies and processes of OSN . Furthermore, we examined the expression of AC3, β-tubulin and OMP by immunofluorescence. We observed significant reductions in the expression of these markers on days 31 and 65 following Zicam administration . In contrast, nasal explants treated with other intranasal agents showed overall healthy morphology with little histological evidence of cytotoxicity .The elevation in extracellular LDH levels following Zicam treatment suggested cell death mediated by necrosis. Histological evaluation of Zicam-treated human nasal tissue indeed confirmed the presence of significant necrosis with the destruction of olfactory epithelium and subepithelial structures . In addition, significant necrosis of the epithelial and subepithelial structures occurred in human nasal explants following Zicam application. Thus, the loss of stem cells is feasible and could explain the lack of recovery of olfactory function in our mouse model. Collectively, the data illustrate that Zicam treatment can damage mouse and human nasal tissue with resultant loss of olfaction.The present study demonstrated that Zicam treatment to both mouse and human nasal tissue induced significant cellular damage. In contrast, treatment with either saline, Afrin, Nasacort, lidocaine and epinephrine did not cause statistically significant tissue damage or olfactory dysfunction. The cytotoxic effects of Zicam were especially profound in the olfactory neuroepithelium. The resultant death of olfactory sensory neurons (OSN) in the MOE contributed to a long-lasting and apparently irreversible olfactory dysfunction in the mouse. Despite the ability of MOE to replace injured OSN via differentiation of stem cells, we found no evidence of functional recovery after two months. In fact, histological analysis of the mouse MOE about two months after Zicam treatment showed complete or near-complete loss of the epithelium and submucosa , and none of these drugs showed any significant signs of cytotoxicity to either mouse or human nasal tissue, confirming their safety profile.Our findings contradict a previous report on the effects of intranasal treatment of Zicam in mice Second, Slotnick and colleagues utilized anterograde tracing with wheat germ agglutinin horseradish peroxidase to determine the integrity of olfactory epithelium ,Many studies have demonstrated the cytotoxic effects of zinc cation on various cell types, including cultured cortical neurons The regenerative capacity of MOE will depend on the extent of necrosis. While our study showed a high degree of cellular damage into deep tissue layers of MOE, variability in clinical presentation  is possible given the variable anatomy of the human nasal cavity. Indeed, published case reports indicate varying outcomes of smell dysfunction associated with Zicam use In this paper, we demonstrated the effects of several commonly used intranasal agents on olfaction using mouse and human nasal tissue. Our study is the most comprehensive examination, to date, of the role that intranasal medications have on short-term and long-term olfactory function. Intranasal administration of Zicam, unlike other tested agents, resulted in significant cytotoxicity to both mouse and human nasal tissue. This is a concerning finding given the potential development of long-lasting, and perhaps irreversible smell dysfunction. Because Zicam was previously classified as a homeopathic substance, it was not required to undergo stringent safety or efficacy evaluation as other conventional drugs. However, the Food and Drug Administration (FDA) recently issued a public health advisory cautioning against the use of some Zicam cold remedy nasal products. The FDA also issued a warning letter to the manufacturers of Zicam reclassifying these products as “drugs” that would require additional safety and efficacy testing to continue to market these products. The potential health risks of intranasal Zicam use demonstrated in our study stresses the need for stringent oversight of homeopathic remedies to protect the public from potentially unknown and dangerous side effects.Adult male C57BL/6 mice  were used for all experiments. All work with animals was approved by the University of Washington Institutional Animal Care and Use Committee.Human subjects were recruited as a consecutive sample from a rhinology clinic at a tertiary medical center. Written consent was obtained from all subjects donating the nasal tissue. Inclusion criteria included any patient over age 18 already scheduled for an endoscopic nasal procedure. Patients were excluded if they had a history of blood borne pathogens. The recruitment of patients and the study protocol was approved by the Institutional Review Board at the University of Washington (#35031).For EOG and biochemical studies, about 15 µl of above intranasal agents were delivered slowly into the right nasal cavity of an adult mouse using a blunted 27-gauge needle. For behavioral testing, about 15 µl of intranasal agent was administered into both nasal cavities using the blunted 27-gauge needle. Animals were sedated with intraperitoneal administration of 80 mg/kg ketamine and 8 mg/kg xylazine prior to intranasal delivery of various agents.2O), mineral oil and dH2O.EOG recordings from the MOE were performed as described previously The odorant habituation assay of adult male mice was performed as described previously Mouse MOE and human nasal tissues were immersed in 4% paraformaldehyde overnight at 4C, followed by cryoprotection in 30% sucrose overnight at 4C. Mouse MOE was decalcified in 0.5 M EGTA at 4C for 3–4 days prior to cryoprotection with 30% sucrose. The tissue was subsequently embedded in OCT , frozen at −20C, cryosectioned at 30 µM and mounted onto slides.Immunofluorescence was performed as described before with the following modifications The tissues were processed as above and air dried for 30 minutes before staining in hematoxylin and eosin  for 1 min each. Tissues were then dehydrated in ascending series of ethanol, cleared in xylene and coverslipped with DPX  and visualized with light microscope.Subject disease severity was assessed with several instruments including a sinus CT scan  2. The culture media was changed every day.The biopsied nasal explants were removed of any tissue debris or blood, and divided into small pieces (approximately 2–4 mm×2–4 mm×1–2 mm) using a 15-blade scalpel and a fine forcep under a dissecting scope. It was then placed on 0.4 µm polytetrafluoroethane (PTFE) membrane  immersed in 1.7 ml of culture medium consisting of 50% Dulbecco's Modified Eagle Medium High Glucose ; 25% Hank solution ; 50 U/mL penicillin G and 40 µg/mL streptomycin  in a 60×15 mm tissue culture dish . The tissue was placed on the membrane such that epithelial side was exposed to the air, and placed in humidified incubator at 37°C with 5% COLDH assay was performed with CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit  with the following modifications. On the day of the assay, 50 µl of culture medium was removed and added to 50 µl of ‘substrate mix’. Following 30 minute incubation at room temperature, the reaction was stopped with 50 µl of ‘stop solution’. Absorbance at 490 nm was obtained with Epx Precision Microplate Reader . For measuring LDH levels in the human nasal explants, we stabilized the nasal tissue in the cultured environment for approximately 48 hours. We then measured the baseline LDH levels, and applied 2 µl of either 0.9% saline, Afrin , Nasacort , lidocaine , 1∶100,000 epinephrine  or Zicam  directly onto the tissue. The levels of LDH were again measured 24 hours later. We repeated the addition of the intranasal agent and LDH measurement for five days.Data are reported as mean +/− S.E.M. We performed statistical analyses using the two-tailed Student t-test. Statistical significance was defined as P<0.05. For the human tissue analysis, a power calculation was performed that showed a sample size of three would give 80% power to detect a significant difference (based on two pilot samples that showed a large effect size). We used linear mixed regression modeling to account for the repeated measures of the LDH assays on successive days for each tissue sample. To account for multiple comparisons, we used Bonferroni adjusted P-values to determine statistical significance (P = 0.05/25 = 0.002 for significance).Figure S1Gross appearance of mouse MOE 9 days after various intranasal agent administrations. Note the atrophy of MOE in Zicam-treated mouse. Black arrowheads indicate atrophic endoturbinates.(3.15 MB TIF)Click here for additional data file.Figure S2Damage to mouse olfactory epithelium following Zicam treatment at various time points. H&E staining of mouse MOE depicting a significant loss of epithelium and submucosal damage 9 days after intranasal administration of Zicam as compared to saline treatment. Much greater damages to the epithelium and submucosal structure are observed without evidence of regeneration 31 and 35 days after intranasal administration of Zicam. Black arrows indicate damaged and remnants of MOE with fibrosis  in Zicam-treated mice. Scale bar, 100 µm.(2.94 MB TIF)Click here for additional data file.Figure S3Cell death in human nasal explants following Zicam treatment from various regions of nasal cavity. H&E staining is shown. (a) Subject 1, inferior turbinate. (b) Subject 2, middle turbinate. (c) Subject 3, superior nasal septum. (d) Subject 4, middle turbinate.(2.87 MB TIF)Click here for additional data file.


Littorina saxatilis. This gastropod displays two sympatric ecotypes (RB and SU) which are becoming one of the best studied systems for ecological speciation.In the past 40 years, there has been increasing acceptance that variation in levels of gene expression represents a major source of evolutionary novelty. Gene expression divergence is therefore likely to be involved in the emergence of incipient species, namely, in a context of adaptive radiation. In this study, a genome-wide expression profiling approach (cDNA-AFLP), validated by quantitative real-time polymerase chain reaction (qPCR) were used to get insights into the role of differential gene expression on the ecological adaptation of the marine snail Among the 99 transcripts shared between ecotypes, 12.12% showed significant differential expression. At least 4% of these transcripts still displayed significant differences after correction for multiple tests, highlighting that gene expression can differ considerably between subpopulations adapted to alternative habitats in the face of gene flow. One of the transcripts identified was Cytochrome c Oxidase subunit I (COI). In addition, 6 possible reference genes were validated to normalize and confirm this result using qPCR. α-Tubulin and histone H3.3 showed the more stable expression levels, being therefore chosen as the best option for normalization. The qPCR analysis confirmed a higher COI expression in SU individuals.At least 4% of the transcriptome studied is being differentially expressed between ecotypes living in alternative habitats, even when gene flow is still substantial between ecotypes. We could identify a candidate transcript of such ecotype differentiation: Cytochrome c Oxidase Subunit I (COI), a mitochondrial gene involved in energy metabolism. Quantitative PCR was used to confirm the differences found in COI and its over-expression in the SU ecotype. Interestingly, COI is involved in the oxidative phosphorylation, suggesting an enhanced mitochondrial gene expression (or increased number of mitochondria) to improve energy supply in the ecotype subjected to the strongest wave action. Unravelling processes that underlie population divergence is a crucial step towards elucidating the origin and maintenance of biodiversity , and towThe development of microarray technology has revolutionized the study of expression profiles since scientists can study tens of thousands of genes at once. However, its utility has been restricted to organisms with extensive cDNA or genomic DNA sequence data available and, of course, any key question in ecology and evolutionary biology cannot be solely addressed using model organisms . A usefuLittorina saxatilis  .On the other hand, quantitative PCR (qPCR) is currently one of the most powerful and sensitive techniques for analyzing gene expression, being often used for validating output data produced by micro-and macro-arrays or other open expression systems. In order to avoid experimental errors arising from variation in the quantity and integrity of the RNA template, as well as the efficiency of the cDNA synthesis and PCR amplification, a normalization step is an essential pre-requisite. Among the several proposed methods ,24, inteLittorina saxatilis involved in an incomplete sympatric ecological speciation process [Therefore, here we will use the cDNA-AFLP technique and qPCR to compare the expression profiles between two ecotypes of  process -45, in oLittorina saxatilis has separate sexes, internal fertilization, and a brood pouch with non-planktonic shelled embryos. In the exposed Galician coast (NO Spain), two well differentiated ecotypes are adapted to different shore levels and habitats [The marine snail habitats ,46. The habitats -49. The habitats -51. Bothhabitats ).Littorina saxatilis ecotypes represent an interesting system to study the genetic basis of adaptive ecological divergence at the transcriptional level in marine species. This work builds upon earlier genome-wide studies showing differences in DNA sequence and protein expression between these ecotypes in the same population [Gene flow between ecotypes is only slightly restricted (8-20%) compared to the level of gene flow among different populations within ecotypes ,52,53; tpulation , allowinMseI-TaqI, with an average of 98.11 ± 1.18 transcripts per primer combination. However, only 99 transcripts were shared in at least 90% of the biological and technical replicates, and therefore considered in the comparison between ecotypes.A total of 16 biological replicates were sampled per ecotype across 2 different transects . Each biological replicate included a pool of 10 adult snails . After total RNA extraction, the quality of the RNA was confirmed spectrophotometrically by the A260/A280 absorbance ratio that ranged from 1.92 to 2.06, indicating a high purity for the RNA extractions. For the cDNA-AFLP analysis, each sample was run twice . Approximately, a total of 2,800 transcripts (75-500 bp long) were obtained from 12 primer combinations using the restriction enzyme pair P < 0.05). Regarding the quantitative data, the averaged Pearson correlation coefficient across samples was used to measure the reproducibility, showing the value of 0.73 , which increased to 0.82 (P < 0.05) when only the transcripts with significant expression differences were taken into account.The coincidence for the presence/absence data was compared by a coefficient of similarity, specifically the "simple-matching coefficient" . Thus, tQuantitative transcript differences between ecotypes were assessed using a three-way ANOVA  on the normalized intensity data (99 transcripts) obtaining 12 transcripts (12.12%) with different level of expression between ecotypes , and 2 of them (numbers 1 and 57) matched with those that were also statistically significant after multitest correction in the three-way ANOVA . This gene seems to be over-expressed in the SU ecotype. Interestingly, this same COI transcript also displayed a differential gene expression between sexes, being higher expressed in males than in females [Six out of 12 transcripts with differential expression between ecotypes showed clear size differences with respect to the other fragments separated in the same AFLP run, making easier their isolation for sequencing purposes. These were therefore isolated and cloned. PCR products obtained from several bacterial colonies containing the insert with the right size were sequenced. All inserts of a particular transcript size were found to be of identical sequence, except for a single heterozygous site in transcript 78  based on the average pairwise variation between all genes tested. Stepwise exclusion of the least stable gene allowed the genes to be ranked according to their M value  . All genThe geNorm program, in addition to the gene stability measure M, computes a normalization factor (NF) and assesses the optimal number of reference genes required for normalization. The pairwise variation (Vn/Vn+1) between two sequential normalization factors (NFn and NFn+1) are used to determine the necessity of adding the next more stable control gene for reliable normalization . PairwisIn order to validate the differential COI expression detected with the cDNA-AFLP technique, primers were designed to amplify COI. The PCR product was isolated and sequenced, confirming its identity Table . Then, qLittorina saxatilis, considered an example of incomplete ecological and sympatric speciation. The experimental reproducibility obtained in this work for the cDNA-AFLP technique is in agreement with previous studies using this technology [Gene expression variation is widespread among individuals and taxa, has a heritable component, and it is subject to influence by natural selection and genetic drift ,57. Therchnology ,59-61. Schnology , we analAlthough the cDNA-AFLP technique is an end-point PCR technique, we note that a crucial characteristic of the AFLP technology is that it is semi-quantitative: the relative intensity of a PCR fragment band in an AFLP fingerprint is related to the original abundance of that fragment in the AFLP template ,64. EvenSimilarly, any putative technical or biological problem associated to this study was resolved by using two complementary types of controls: First, using both biological and technical replication and showing differences in the biological replicates despite of the detected technical noise. Second, gene expression differences between ecotypes were compared with those observed within ecotypes. So, any possible biases affecting our study should affect similarly to the within-and between-ecotype differentiation, while our results showed that significant transcript differences were observed exclusively between ecotypes.The observed differences in gene expression could be heritable, caused by environmental effects linked to the different habitats associated to each ecotype, or result from a combination of environmental and heritable factors. Nevertheless, a recent study has shown that much of the variation in mRNA expression is related to genetic variation, and that only a minor part of this variation occurs in response to environmental changes . SimilarSorting or clustering genes into groups according to their expression patterns can provide a broad and interpretable overview of gene regulation . Here, wRegarding the identification of the transcripts, most of the differentially expressed genes did not correspond to known sequences in the available databases. This could be due to the scarcity of gastropod sequences in public databases or, alternatively, these sequences may represent novel mRNAs. Another possibility is that they could correspond to the 3' untranslated region of the gene where the sequences are often less conserved than the sequences of protein-coding regions . HoweverL. saxatilis, we decided to discard β-actin gene, one of the most traditionally used in qPCR, for two main reasons: i) both ecotypes show differences in musculature ; ii) itd muscle ,74. Accod muscle . In thisper se. Previous studies showed that higher levels of mtDNA gene products under physiological stimuli are primarily met by mtDNA replication  confirmed the results obtained previously by cDNA-AFLP. This up-regulation of the COI in the SU ecotype could result both from an increased number of mitochondria in the SU ecotype and/or changes in transcription rate iewed in ). Cytochiewed in . COX is iewed in . Changesiewed in . As COX iewed in , where eiewed in . The SU iewed in . Therefoiewed in ,78. Furtous work . A similunctions ,80. Thisunctions ,81-84. Tunctions ) is muchunctions ,86.A major challenge towards a comprehensive analysis of speciation processes is the integration of data from different "omics" resources and their interpretation. Even when we are very cautious and taking all the precautions due to the inherent differences caused by the distinct techniques, we could make a proxy of what is happening at three different biological levels. At genome level, both ecotypes differed around 3% in their genome by an AFLP scan , whereasAnother central, and yet controversial, question in evolutionary biology concerns the genetic basis of evolutionary change. King and Wilson (1975)  proposedLittorina saxatilis. Our results show that at least 4% of the sampled transcripts are being differentially expressed between ecotypes adapted to alternative habitats, highlighting important differences in gene expression in the face of gene flow. A candidate transcript of such ecotype differentiation was identified as Cytochrome c Oxidase Subunit I (COI), which is up-regulated in the exposed ecotype. Interestingly, COI is involved in the oxidative phosphorylation, suggesting an enhanced mitochondrial gene expression (or increased number of mitochondria) to improve energy supply in the ecotype subjected to the strongest wave action, in the same way that those genes identified at proteome level [In this study we have improved our knowledge of the role of differential gene expression on the ecological adaptation of the marine snail me level .L. saxatilis were collected from Silleiro  in two different transects. RB and SU individuals were collected from the upper and lower shore levels of each transect respectively. The horizontal distance between the two transects was 50 m, whereas the vertical distance between the two ends of each transect was approximately 23 m. Eight biological replicates were sampled per shore level . Samples were collected simultaneously during 30 minutes and immediately transported to the laboratory, where snails were sexed according to the presence of penis in males and a brood pouch of shelled embryos in females, thus ensuring that individuals were sexually mature. Each biological replicate (of the RB or SU ecotype) contained a pool of 5 males and 5 females. Snails used for the qPCR analysis were collected from Silleiro in July 2009 using the same procedure. However, in this case, 9 biological replicates were used for each ecotype, each one including a pool of 5 males and 5 females.In July 2006, adult males and females of ® Reagent according to the manufacture's instructions. High quality starting RNA is essential for the cDNA-AFLP technique. Therefore, in order to assess the integrity of the total RNA, an aliquot of each sample was run on an agarose gel. Moreover, the concentration and purity (i.e. the A260/A280 ratio) of each RNA sample was checked with an UV spectrophotometer (UNICAM UV/Vis UV2). Next, the Turbo DNA-freeTM kit (Ambion) was used to remove any remaining contaminating DNA from the total RNA extractions. The concentration was measured again by spectrophotometry. Finally, cDNA was synthesized from 25 μg of total RNA using the SuperScriptTM Double-Stranded cDNA Synthesis Kit (Invitrogen) and a biotinylated oligo (dT)18 primer, following the conditions recommended by the manufacturer.Prior to RNA extraction, shelled embryos of each female were discarded. Total RNA was isolated from pools containing 5 males and 5 females of the RB or SU ecotype using TRIZOLet al. (2010)[TaqI and MseI, each with a 4 bp recognition sequence to obtain enough number of peaks by primer combination as in previous works with cDNA [TaqI for 4 h at 37°C. Then, the enzyme was inactivated by heating for 10 min at 70°C and the 3' end fragments were collected on streptavidin magnetic beads by virtue of their biotinylated tails using the PolyATract® Systems III kit (Promega). Next, cDNA fragments on the magnetic beads were digested with MseI for 4 h at 37°C, followed by the inactivation of the enzyme for 4 h at 37°C. The supernatant, containing the digested fragments, was collected and used as template in the subsequent AFLP steps, while the 3'-end tails that remained bounded to the beads were discarded. In this way, the total number of tags to be screened was reduced, additionally favouring the collection of more informative tag derived at least partially from the coding region, and therefore facilitating functional characterization of the transcripts. MseI and TaqI adaptors were ligated for 16 h at 16°C in a total volume of 50 μl. The MseI and TaqI adaptors were prepared by mixing equimolar amounts (50pmol each) of the oligonucleotides 5'-GACGATGAGTCCTGAG-3' and 5'-TACTCAGGACTCAT-3' for MseI adaptor and 5'-GACGATGAGTCCTGAG-3' and 5'-CGCTCAGGACTCAT-3' for TaqI adaptor. Pre-amplification of cDNA fragments was performed for 20 cycles with a 4 μl aliquot of a 1:10 dilution of the ligation reaction in a total volume of 20 μl, using 20 pmol of the primers corresponding to the MseI and TaqI adaptor sequence without selective base . Each PCR was performed at 94°C for 20 sec, 56°C for 30 sec, and 72°C for 2 min. The pre-amplified products were diluted 1:10 and a 4 μl aliquot was selectively amplified in a total volume of 20 μl with MseI and TaqI primers having 1 selective base extension at the 3' end. Amplification included a touchdown phase for 10 cycles of PCR , followed by 20 cycles . A total of 12 primer combinations were used for selective amplifications. TaqI selective primers were fluorescently labeled with different dyes . The selectively amplified fragments were run on an ABI Prism 310 Genetic Analyzer with an internal ROX-labelled sizing ladder (Applied Biosystems).The cDNA-AFLP technique  was usedl. (2010). The resith cDNA ,94. The ® v.3.7 software (Applied Biosystems), within a fragment-length (size) range of 75-500 base pairs. To eliminate background noise, a DNA fragment was considered to be valid if it had a peak height of at least 50 relative fluorescent units (RFU) and a ± 2 base size difference with the nearest DNA fragment peak (transcript). Each AFLP expression profile was normalized using the sum of signal method as implemented in GeneMapper® to correct for differences in total electropherogram intensities, which may arise due to loading errors or differences in amplification efficiency, being actually a semi-quantitative analysis. Moreover, all AFLP reactions were repeated twice  to allow evaluation of the reproducibility of the method, which was calculated by the Pearson correlation coefficient for the quantitative data, and by the coefficient of similarity called "simple-matching coefficient" [AFLP profiles were visualized and analyzed using GeneMapperficient"  for the ficient" .We used the whole set of 32 samples  to choose exclusively those transcripts that were present in 90% of the replicates. In the randomization ANOVA, when a transcript was only detected in one of the two technical replicates, we assigned the threshold limit value of 50 RFU to the missing peak height, and used the average value of the two technical replicates.Only common transcripts (present in at least 90% of the replicates) were considered in the comparison among groups. Quantitative transcript differences were assessed using a three-way ANOVA on normalized intensity data with the two fixed factors (ecotype and transect), the third factor  nested with the interaction, and the technical replicates as residual. This parametric three-way ANOVA was carried out with the SPSS/PC version 14. Here, the factor transect could be considered as a control of expression differences between ecotypes in the sense that any putative candidate loci of adaptation should produce significant differences in expression between ecotypes, but not significant differentiation between transects or interaction.http://webs.uvigo.es/c03/webc03/XENETICA/XB2/software.htm, which is very robust to deviations from normality and heteroscedasticity . The SGity . To reamplify the differentially expressed transcripts, 1 μl of the eluted cDNA was amplified with the same selective primer combination in a 20 μl reaction volume. PCR conditions were an initial denaturation step at 95°C for 5 min, 30 cycles at 95°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec, with a final extension step at 72°C for 10 min. The size of the amplified fragments was confirmed by capillary electrophoresis on an ABI Prism 310 Genetic Analyzer in order to test whether the isolated peak showed the expected size. The PCR product was purified and cloned using TOPO TA Cloning® kit for Sequencing (Invitrogen). Finally several colonies from each PCR product were sequenced in order to determine if a single transcript had been effectively isolated from each peak, or whether if a peak contained more than one transcript type (homoplasy). Nucleotide sequences and translated sequences were compared with the nucleotide and protein sequences of the nonredundant Genbank databases and the sequences of the EST databases using BLAST and TBLAST sequence alignment programs respectively at the web server of NCBI in july 2010.In order to isolate the peaks showing significant differences in expression, the PCR products of the corresponding selective primer combination were run in agarose gels to cut the bands corresponding to the appropriate peak size. cDNA was extracted from the gel piece using NucleoSpin® Reagent  according to the manufacture's instructions. The integrity of the total RNA was assessed in the same way than for the cDNA-AFLP analysis. Genomic DNA was eliminated from the samples by a DNase treatment (Fermentas) according to the manufacturer's description, and its concentration was again determined by spectrophotometry. RNA from each sample (1 μg) was reverse transcribed in a final volume of 20 μl using the Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science) and an oligo (dT)18 primer.For the qPCR analyses, total RNA was isolated from the pools using TRIZOLhttp://frodo.wi.mit.edu/primer3/. To ensure that fluorescent signals produced during qPCR assays represent only target amplicons, primer conditions were previously optimized by determining the optimal annealing temperature and primer concentrations to eliminate the formation of primer dimers that could contribute to fluorescence. Specificity of the amplicons was checked by running the conventional PCR products on 2% agarose gel to confirm the production of a single band of the predicted size. Moreover, a melting curve analysis was performed after every amplification program in the qPCR to verify specificity of the target and absence of primer dimers, besides of including a no-template control on every plate for each primer set to verify also that PCR master mixes were free of contamination.Specific primer pairs were designed for 6 candidate reference genes and 1 for the gene of interest Cytochrome c Oxidase subunit I (COI). Primer design was done using Primer 3  with 20 μl reactions containing 10 μl of LightCyclern480 SYBR Green I Master, 7.6 μl or RNase free water, 0.2 μl of each primer (1 μM), and 2 μl of cDNA as PCR template. Cycling parameters were 95°C for 10 min to activate DNA polymerase followed by 35 cycles of 95°C for 10 s, 54°C for 15 s, and a final extension of 72°C for 9 s. Detection fluorescence was carried out at the end of each extension step. After amplification, a melting curve was acquired by heating to 95°C for 5 s, cooling to 70°C for 1 min, and slowly heating to 95°C with a continuous fluorescence data collection of 10 acquisitions per °C.To determine the mRNA expression, stability of the 6 reference genes was measured using the qPCR as described above. Since the use of just a single reference gene may result in a more than 6-fold erroneous normalization , the geNq) values were transformed to relative quantities using the software program geNorm, version 3.4 [http://medgen.ugent.be/~jvdesomp/genorm/. The statistical algorithm geNorm derives a stability measure  and via a stepwise exclusion of the least stable gene, creates a stability ranking. Genes with the lowest M values have the most stable expression, and therefore would be selected as ideal reference genes. In the iterative steps, genes with the lowest expression stability  are removed. A new M value for each of the remaining genes is calculated until only two genes remain. Because these calculations are based on ratios, the final 2 genes cannot be resolved.Raw quantification cycle .In order to determine the differential expression of the gene of interest (COI), raw Cq values were transformed to relative quantities, normalized by the two best reference genes, using the software qBase version 1.2.2 (based on the formulas described in ). Then, MMF: carried out the molecular genetic studies, participated in the sequence alignment, drafted the manuscript, participated in the design of the study, and performed the statistical analysis; LB: participated in the study design and coordination, and helped to draft the manuscript; ERA & HQ: participated in the design of the study, performed the statistical analysis, conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.