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Results for “single cell integration”

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PubMed1996

Mutagenesis of AS52 cells by low concentrations of lead(II) and mercury(II)

Little is known at the molecular level concerning the genotoxic effects following the acute exposure of eukaryotic cells to low concentrations of lead (II) or mercury (II). There have been conflicting reports concerning the mutagenic potential of these heavy metals, and there have not been any studies performed to determine the molecular mechanism(s) by which these metals are mutagenic. The Chinese hamster ovary cell line, AS52, contains a stably integrated single functional copy of the Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene confer resistance to 6-thioguanine (TG). There was little effect on viability, as measured by relative cloning efficiency, of AS52 cells exposed to lead (II) or mercury (II) up to concentrations of 0.5 microM and 0.3 microM, respectively. However, higher concentrations of the metals caused a significant increase in cell death. There was also a dose-dependent increase in the isolation of mutants resistant to TG in treated cells when compared to non-treated controls. Concentrations of the metals as low as 0.1 microM caused a significant increase in the number of mutants resistant to TG when compared to the number of spontaneous mutants obtained in nontreated controls. While the molecular mechanism(s) by which lead and mercury (II) are genotoxic is unknown, the results of this study demonstrate that low concentrations of lead (II) and mercury (II) are mutagenic in eukaryotic cells.

Animals↗

PubMed2026

A machine learning-derived and functionally validated circadian rhythm signature predicts clinical outcomes and in silico drug sensitivity in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) displays considerable heterogeneity in clinical outcomes, highlighting the need for reliable prognostic biomarkers. While the aberrant expression of circadian rhythm-related genes has been implicated in cancer pathogenesis, its comprehensive role in CRC progression and predicted therapeutic vulnerabilities remains inadequately characterized. METHODS: Bulk and single-cell RNA-sequencing data were integrated from multiple CRC cohorts. A circadian rhythm signature (CRS) was developed through machine learning algorithms and validated for prognostic value. Comprehensive analyses of tumor microenvironment, genomic alterations, and drug sensitivity were performed. Furthermore, the biological function of the core gene, BHLHE40, was validated in CRC cell lines through CCK-8, EdU, and wound healing assays. RESULTS: Single-cell analysis demonstrated an elevated expression signature of circadian rhythm-related genes in dendritic cells. The optimized CRS, comprising 14 circadian rhythm-related genes, successfully categorized patients into high- and low-risk groups. Patients with a high CRS showed markedly poorer overall survival and computationally inferred immunosuppressive features, including reduced CD8+ T cell infiltration and increased M2 macrophage polarization. Genomic analysis revealed enhanced mutation burden in TP53 and alterations in RTK-RAS/WNT pathways. Notably, in vitro assays confirmed that BHLHE40 is significantly overexpressed in CRC cells. Knockdown of BHLHE40 markedly inhibited tumor cell proliferation and migration. Drug sensitivity profiling identified bexarotene and SMER-3 as potential therapeutic options for high-CRS patients. A nomogram integrating CRS with clinical parameters demonstrated superior predictive accuracy for 1-, 3-, and 5-year survival. CONCLUSIONS: The CRS represents a promising prognostic biomarker that reflects tumor immune status and genomic features, providing valuable insights for personalized treatment strategies in CRC.

Circadian rhythm↗

PubMed2006

Integration of auditory and visual communication information in the primate ventrolateral prefrontal cortex.

The integration of auditory and visual stimuli is crucial for recognizing objects, communicating effectively, and navigating through our complex world. Although the frontal lobes are involved in memory, communication, and language, there has been no evidence that the integration of communication information occurs at the single-cell level in the frontal lobes. Here, we show that neurons in the macaque ventrolateral prefrontal cortex (VLPFC) integrate audiovisual communication stimuli. The multisensory interactions included both enhancement and suppression of a predominantly auditory or a predominantly visual response, although multisensory suppression was the more common mode of response. The multisensory neurons were distributed across the VLPFC and within previously identified unimodal auditory and visual regions (O'Scalaidhe et al., 1997; Romanski and Goldman-Rakic, 2002). Thus, our study demonstrates, for the first time, that single prefrontal neurons integrate communication information from the auditory and visual domains, suggesting that these neurons are an important node in the cortical network responsible for communication.

Acoustic Stimulation↗

PubMed1991

When Epstein-Barr virus persistently infects B-cell lines, it frequently integrates.

In this study we used Gardella gel analysis of intact DNA, Southern blotting of digested DNA, and fluorescence in situ hybridization to provide complementary and unequivocal information on the state of the Epstein-Barr virus (EBV) genome in persistently infected cells. The fluorescence in situ hybridization technique allowed us to directly visualize both integrated and episomal EBV DNA at the single-cell level. We show here that circularization of the EBV genome is rarely detected upon infecting activated normal B cells. The virus can persist upon infection of a different proliferating B-cell target, EBV-negative Burkitt's lymphoma tumor cell lines. Analysis of 16 such lines reveal again, that the virus infrequently persists as covalently closed episomes; rather, the virus preferentially persists by integrating into the host DNA (10 of 16 clones). The integrated virus is linear and usually intact, although 3 of 10 isolates have deletions from the left-hand end including the latent origin of replication. At the level of our analysis, no obvious relationship was seen between the integration sites. These studies provide, for the first time, a reproducible in vitro model system to study integration by EBV.

B-Lymphocytes↗

PubMed1989

Murine cell lines stably expressing the influenza virus hemagglutinin gene introduced by a recombinant retrovirus vector are constitutive targets for MHC class I- and class II-restricted T lymphocytes.

A retrovirus vector containing the hemagglutinin (HA) gene of influenza virus was constructed and used to infect murine cell lines of fibroblast, mastocytoma and B cell lineages which are able to present antigens to MHC-restricted T cells. Stable cell lines were selected in which the retrovirus vector integrated as a single copy in almost all of the individual cell clones examined. The HA mRNA was shown to be of the expected length by Northern blot analysis, but the levels varied among the cell clones. Although the HA transcript was difficult to detect in any of the retrovirus-infected cell clones derived from fibroblasts, HA Ag was easily detected on the cell surface by cytofluorographic analysis. Significantly, retrovirus-infected clones derived from each cell type were recognized by HA-specific class I and class II MHC-restricted T lymphocytes. HA produced in these cells was able to be acquired, processed, and presented to class II-restricted T cells by additional, non-HA-expressing APC. This indicates that HA endogenously synthesized within these cell lines is available for Ag processing by an exogenous route.

Animals↗

PubMed2006

Selection of cervical keratinocytes containing integrated HPV16 associates with episome loss and an endogenous antiviral response.

Integration of high-risk human papillomavirus (HRHPV) into the host genome is a key event in cervical neoplastic progression. Integration is associated with deregulated expression of the viral oncogenes E6 and E7 and acquisition of a selective growth advantage for cells containing integrants. Overexpression of the viral transcriptional regulator E2 from heterologous promoters has an inhibitory effect on transcription from integrated HRHPV. Therefore, we hypothesized that loss of E2-expressing episomes from cells in which integration had previously occurred would be required for such cells to gain a growth advantage. Using the unique W12 model of cervical squamous carcinogenesis, we show that cells containing integrated HPV16 reproducibly emerged during long-term culture when there had been a rapid fall in episome numbers. During the period of emergence, it is possible to isolate single-cell clones containing an intracellular mixture of the integrant being selected and episomes at reduced load. The lower level of E2 expression seen in such cells is associated with partial inhibition of transcription from the HPV16 integrant. Full deregulation is not observed until complete loss of E2-expressing episomes occurs. Microarray analysis showed that episome loss was closely associated with endogenous activation of antiviral response genes that are also inducible by the type I IFN pathway. Taken together, our results indicate that episome loss, associated with induction of antiviral response genes, is a key event in the spontaneous selection of cervical keratinocytes containing integrated HPV16. We conclude that cervical carcinogenesis requires not only HRHPV integration, but also loss of inhibitory episomes.

Cell Line↗

PubMed1998

Single-cell measurements of purine release using a micromachined electroanalytical sensor.

To study the cellular events surrounding the formation of purines in cardiac ischemia, we have micromachined a micrometer-scale titer chamber containing an integrated electrochemical sensor, capable of measuring analytes produced by a single heart cell. The analytical procedure involves the determination of metabolites via the amperometric detection of enzymically generated hydrogen peroxide, measured at a platinized microelectrode, poised at a suitably oxidizing potential, equivalent to +420 mV vs Ag/AgCl. Signals were recorded as current-time responses and were integrated to give a total charge (Q) attributable to the reaction under investigation. The amount of analyte produced by the cell was subsequently quantified by the addition of a known amount of calibrant. As a consequence, by using a cascade of three enzymes (adenosine deaminase, nucleotide phosphorylase, and xanthine oxidase), we were able to show that, after rigor contracture had been induced in a single myocyte, adenosine (but not inosine) only reached the extracellular space after the cell membrane had been permeabilized by detergent. These data, which could only be obtained unambiguously by using this single-cell methodology, have provided us with information on the origin of ischemic adenosine which challenges the established assumption that purine release is an early retaliatory response from intact anoxic myocytes.

Biosensing Techniques↗

PubMed2025

scPlantLLM: A Foundation Model for Exploring Single-cell Expression Atlases in Plants.

Single-cell RNA sequencing (scRNA-seq) provides unprecedented insights into plant cellular diversity by enabling high-resolution analyses of gene expression at the single-cell level. However, the complexity of scRNA-seq data, including challenges in batch integration, cell type annotation, and gene regulatory network (GRN) inference, demands advanced computational approaches. To address these challenges, we developed scPlantLLM, a Transformer model trained on millions of plant single-cell data points. Using a sequential pretraining strategy incorporating masked language modeling and cell type annotation tasks, scPlantLLM generates robust and interpretable single-cell data embeddings. When applied to Arabidopsis thaliana datasets, scPlantLLM excels in clustering, cell type annotation, and batch integration, achieving an accuracy of up to 0.91 in zero-shot learning scenarios. Furthermore, the model demonstrates an ability to identify biologically meaningful GRNs and subtle cellular subtypes, showcasing its potential to advance plant biology research. Compared to traditional methods, scPlantLLM outperforms in key metrics such as adjusted rand index (ARI), normalized mutual information (NMI), and silhouette score (SIL), highlighting its superior clustering accuracy and biological relevance. scPlantLLM represents a foundation model for exploring plant single-cell expression atlases, offering unprecedented capabilities to resolve cellular heterogeneity and regulatory dynamics across diverse plant systems. The code used in this study is available at https://github.com/compbioNJU/scPlantLLM.

Single-Cell Analysis↗

PubMed2002

Direct ex vivo analysis of human CD4(+) memory T cell activation requirements at the single clonotype level.

CD4(+) memory T cells continuously integrate signals transmitted through the TCR and costimulatory molecules, only responding when the intensity of such signals exceeds an intrinsic activation threshold. Recent data suggest that these activation thresholds can be regulated independently of TCR specificity, and that threshold tuning may constitute a major mechanism for controlling T cell effector activity. In this work we take advantage of the profound clonotypic hierarchies of the large human CD4(+) T cell response to CMV to study activation thresholds of fresh (unexpanded) memory T cells at the clonotypic level. We identified dominant responses to CMV matrix determinants mediated by single TCRB sequences within particular TCR-Vbeta families. The specific response characteristics of these single, Ag-specific, TCRB-defined clonotypes could be unequivocally determined in fresh PBMC preparations by cytokine flow cytometry with gating on the appropriate Vbeta family. These analyses revealed 1) optimal peptides capable of eliciting specific responses by themselves at doses as low as 2 pg/ml, with each log increase in dose eliciting ever-increasing frequencies of responding cells over a 4- to 5-log range; 2) significant augmentation of response frequencies at all submaximal peptide doses by CD28- and CD49d-mediated costimulation; 3) differential dose response and costimulatory characteristics for IFN-gamma and IL-2 responses; and 4) no association of activation requirements with the CD27-defined CD4(+) T cell memory differentiation pathway. Taken together these data confirm that triggering heterogeneity exists within individual CD4(+) memory T cell clonotypes in vivo and demonstrate that such single clonotypes can manifest qualitatively different functional responses depending on epitope dose and relative levels of costimulation.

Antigen-Presenting Cells↗

PubMed1997

Different integrated and unintegrated HIV-1 DNA after superinfection and cell to cell transmission.

Efficient superinfection of H9HTLVIIIB cell line (persistently infected with HIVHXB2 strain) with HIVMN strain is reported. The superinfecting viral DNA was found in the chromosomic and extrachromosomic fractions at early stages, but at 48 hours post superinfection, it remained mainly unintegrated. Interestingly, superinfected cells only produced HIVHXB2 in the supernatant and no increase of viral yield of this persistent virus was observed. Remarkably, virions of both strains. HIVHXB2 and HIVMN, were recovered after cocultivating superinfected cells with MT2 cell line. In the extrachromosomic fractions of seven different superinfected subclons of H9HTLVIIIB, viral DNA of the superinfecting HIVMN strain predominated while in the chromosomic fraction, the proportion of superinfecting viral DNA differed. The study of the presence of different integrated and unintegrated genomes in a single cell could be crucial in the understanding of HIV biology.

Cell Line↗

PubMed2002

A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye.

Regulated cell death and survival play important roles in neural development. Extracellular signals are presumed to regulate seven apparent caspases to determine the final structure of the nervous system. In the eye, the EGF receptor, Notch, and intact primary pigment and cone cells have been implicated in survival or death signals. An antibody raised against a peptide from human caspase 3 was used to investigate how extracellular signals controlled spatial patterning of cell death. The antibody crossreacted specifically with dying Drosophila cells and labelled the activated effector caspase Drice. It was found that the initiator caspase Dronc and the proapoptotic gene head involution defective were important for activation in vivo. Dronc may play roles in dying cells in addition to activating downstream effector caspases. Epistasis experiments ordered EGF receptor, Notch, and primary pigment and cone cells into a single pathway that affected caspase activity in pupal retina through hid and Inhibitor of Apoptosis Proteins. None of these extracellular signals appeared to act by initiating caspase activation independently of hid. Taken together, these findings indicate that in eye development spatial regulation of cell death and survival is integrated through a single intracellular pathway.

Amino Acid Sequence↗

PubMed2026

Site-specific gene integration by recombinase-mediated cassette exchange in anhydrobiotic Pv11 cells.

Pv11 cells, derived from Polypedilum vanderplanki, uniquely tolerate complete desiccation (anhydrobiosis). Although a CRISPR/Cas9-based precise integration method (CRIS-PITCh) has been developed for Pv11 cells, a CRISPR-free strategy that fixes both the genomic locus and transgene copy number has not yet been established. Here, we implement recombinase-mediated cassette exchange (RMCE) in Pv11 cells and generate master cell lines that retain anhydrobiosis following genetic engineering. We first evaluated the activity of multiple site-specific recombinases in Pv11 cells using a transient two-plasmid reporter assay. Flp, Cre, and Bxb1 recombinases all excised a test cassette and activated a green fluorescent protein (GFP) reporter, whereas phiC31 integrase mediated recombination at the DNA sequence level but did not induce reporter expression under our construct configuration. To enable genomic RMCE, we inserted an FRT/FRT3-landing pad (LP) into a previously identified genomic safe-harbor locus using CRIS-PITCh and isolated clonal master cell lines by single-cell sorting. Using the established master line, Flp-based RMCE achieved site-specific cassette exchange at the LP, producing HaloTag fluorescence and drug resistance upon successful exchange. In addition, the expected post-exchange sequence was confirmed by sequencing. We further established an all-in-one RMCE vector combining the Flp recombinase and donor cassette on a single plasmid. Together, these results demonstrate locus-defined, single-copy transgene integration in anhydrobiotic Pv11 cells via RMCE and provide a standardized, CRISPR-free workflow for routine genetic manipulation in this unique cell line. This workflow facilitates both fundamental research and applied biotechnological applications using desiccation-tolerant cells.

Anhydrobiosis↗

PubMed2006

A risk score for mortality after allogeneic hematopoietic cell transplantation.

BACKGROUND: Despite recent advances, mortality rates after allogeneic hematopoietic cell transplantation remain high and cannot be accurately predicted. OBJECTIVE: To develop a reliable and valid predictor of all-cause mortality during the first 2 years after allogeneic hematopoietic cell transplantation. DESIGN: Retrospective cohort. SETTING: Tertiary hematopoietic cell transplantation center. PATIENTS: Patients (n = 2802) who received a first hematopoietic cell transplant between 1990 and 2002 were assigned to a development group or a validation group. MEASUREMENTS: Potential predictor variables were assessed with univariate and multivariable Cox proportional hazards methods to generate a prediction model. The c-statistic was calculated for 5 validation cohorts to assess model performance across early and late time periods and among patients with different diagnoses. RESULTS: The authors constructed a 50-point Pretransplantation Assessment of Mortality (PAM) score that incorporated 8 pretransplantation clinical variables: patient age, donor type, disease risk, conditioning regimen, FEV1, carbon monoxide diffusion capacity, serum creatinine level, and serum alanine aminotransferase concentration. The risk for death within 2 years for patients with PAM scores in the highest category was significantly higher than for those with scores in the lowest category. C-statistic values ranged from 0.69 to 0.76 for all validation cohorts. LIMITATIONS: The predictor model was not validated in an external cohort and is only useful for predicting the risk for death within the first 2 years after hematopoietic cell transplantation. CONCLUSIONS: Integrating pretransplantation clinical variables into a single score reliably predicts survival within 2 years after allogeneic hematopoietic cell transplantation. Accurate estimates of the risk for death may be useful in clinical trials and in epidemiologic studies. Such information can also be used to help physicians counsel patients regarding the expected outcomes of this potentially curative procedure.

Adult↗

PubMed1985

Genetic analysis of integration mediated by single T-DNA borders.

Transformation of plant cells by the T-DNA of the Ti plasmid of Agrobacterium tumefaciens depends in part upon a sequence adjacent to the right T-DNA end. When this sequence is absent, the T-DNA is almost avirulent; when it is present, DNA between it and the left T-DNA border region becomes integrated in plants. To investigate further this process of DNA transfer and integration, we introduced the right border region and the nopaline synthase (nos) gene of plasmid pTiC58 into a variety of new positions around Ti plasmids. The border region functioned when separated from the remainder of the T-DNA by almost 50 kilobases. It also worked when placed outside of the T-DNA region where there were no known left-border sequences with which to interact. Indeed, the nos gene could be transferred to plants even when no other Ti plasmid sequences were present on the same plasmid. These results may indicate that the sequence requirements for the left borders are not as stringent as those for the right borders. In addition, mutants with an extra copy of the right border region within their T-DNA were found to transfer or integrate only parts of the bacterial T-DNA region. It is possible that abnormally placed T-DNA borders interfere with the normal process of DNA transfer, integration, or both.

Base Sequence↗

PubMed2006

Metabolic monitoring of the electrically stimulated single heart cell within a microfluidic platform.

A device based on five individually addressable microelectrodes, fully integrated within a microfluidic system, has been fabricated to enable the real-time measurement of ionic and metabolic fluxes from electrically active, beating single heart cells. The electrode array comprised one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an electrochemical lactate microbiosensor, that were used to measure the amounts of lactate produced by the heart cell. The device also allowed simultaneous in-situ microscopy, enabling optical measurements of cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+. Initial experiments aimed to create a metabolic profile of the beating heart cell, and results show well defined excitation-contraction (EC) coupling at different rates. Ca2+ transients and extracellular pH measurements were obtained from continually paced single myocytes, both as a function of the rate of cell contraction. Finally, the relative amounts of intra- and extra-cellular lactate produced during field stimulation were determined, using cell electroporation where necessary.

Aerobiosis↗

PubMed1995

Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA.

Primary human vascular endothelial cells were immortalized by the integration of a single DNA copy of an amphotrophic, replication-deficient retrovirus containing the E6/E7 genes of human papilloma virus. To date, the resulting cell lines, designated EC-RF7 and EC-RF24, have been cultured for more than 1 year. The cell lines have retained a diploid karyotype, display no abnormalities, and are able to grow in a polar mode. Analysis of the EC-RF cell lines by indirect immunofluorescence, using an extensive panel of monoclonal antibodies, showed expression of endothelial cell-specific soluble (von Willebrand factor) and surface-bound antigens (endoglin, PCAM-1) indistinguishable from that of primary cells. In addition, the expression of the markers CD9, 13, 14, 29, 36, 40, 51, and 55 that are not restricted to endothelial cells was also similar for the immortalized and the primary endothelial cells. Immortalization did not alter the expression of the surface adhesion molecules E-selectin, VCAM-1, and ICAM-1 nor transmigration of neutrophils. The regulation of extracellular proteolytic activity by EC-RF24 was established by measuring both the induction of functional tissue factor (promotion of Factor Xa generation) and the functional deposition of plasminogen activator inhibitor 1 in the subendothelial matrix (SDS-resistant complex formation with thrombin). Finally, the biosynthesis of the endothelial cell-specific von Willebrand factor was studied in detail in the EC-RF24 cell line and the results were compared with those of primary endothelial cells.

Antigens, CD↗

PubMed1999

The effect of ascorbate and alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa.

The aim of this study was to determine the effects of supplementation with ascorbate and alpha-tocopherol, both singly and in combination, during sperm preparation on subsequent sperm DNA integrity, induced DNA damage and reactive oxygen species (ROS) generation. Semen samples with normozoospermic and asthenozoospermic profiles (n = 15 for each control and antioxidant group) were prepared by Percoll density centrifugation (95.0-47.5%) where the medium had been supplemented with these antioxidants to a number of different concentrations, all within physiological levels. Controls were included which had no ascorbate or alpha-tocopherol added. DNA damage was induced using hydrogen peroxide (H(2)O(2)) and DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay, while ROS generation was measured using chemiluminescence. Addition of ascorbate to sperm preparation medium did not affect baseline DNA integrity but did provide sperm with complete protection against H(2)O(2)-induced DNA damage. Generation of H(2)O(2)-induced ROS was also significantly reduced after treatment with ascorbate, although baseline levels were unaffected by this antioxidant. Supplementation of sperm preparation medium with alpha-tocopherol did not influence baseline DNA integrity but provided sperm with dose-dependent protection against H(2)O(2)-induced DNA damage. Generation of H(2)O(2)-induced ROS was significantly reduced after treatment with alpha-tocopherol, although baseline ROS levels were unaffected by this antioxidant. Addition of both ascorbate and alpha-tocopherol in combination to sperm preparation medium actually induced DNA damage and intensified the damage induced by H(2)O(2), however, H(2)O(2)-induced ROS production was significantly reduced in a dose-dependent manner by supplementation with both vitamins.

Antioxidants↗

PubMed2000

Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa.

Sperm DNA integrity is essential for accurate transmission of genetic material to offspring. Fragmentation of genomic DNA is an initial hallmark of apoptosis (programmed cell death). The aim of this study was to determine sperm nuclear DNA integrity and mitochondrial function, to quantify possible apoptosis and to investigate any relationship between these parameters. Semen samples (n = 25) were prepared by discontinuous Percoll density centrifugation (95.0:47.5). DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly indicative of apoptosis, was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Mitochondrial transmembrane potential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The DNA integrity of prepared spermatozoa was significantly greater than that of semen (P < 0.005). Further, the percentage of spermatozoa with fragmented DNA and the degree of fragmentation within these cells in prepared spermatozoa is significantly less than in semen (P < 0.005). There is a significant correlation between DNA damage quantified using the Comet assay and DNA fragmentation determined using TUNEL (R = 0.562, P < 0.01). The percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria was significantly lower in prepared spermatozoa than in neat semen samples (P < 0.001). There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile spermatozoa (R = -0.67, P < 0.01).

Adult↗