Flow cytometric evaluation of sperm parameters in relation to fertility potential.

Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used in the future to monitor many new potential markers of sperm function.

[1]  R. Foote.  Fertility estimation: a review of past experience and future prospects. , 2003, Animal reproduction science.

[2]  C. Love,et al.  The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion. , 1998, Theriogenology.

[3]  H. Woelders,et al.  Enhanced binding of zona pellucida proteins to the acrosomal region of the intact boar spermatozoa in response to fertilizing conditions: a flow cytometric study. , 1998, Biology of reproduction.

[4]  B. Gadella,et al.  Evaluation of In Vitro Capacitation of Stallion Spermatozoa1 , 2001, Biology of reproduction.

[5]  S. Oehninger,et al.  Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. , 2002, Human reproduction.

[6]  Serafino Pantano,et al.  Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. , 1995, Biology of reproduction.

[7]  H. Rodríguez-Martínez,et al.  Can we use in vitro fertilization tests to predict semen fertility? , 2000, Animal reproduction science.

[8]  D. Donoghue,et al.  Viability assessment of turkey sperm using fluorescent staining and flow cytometry. , 1995, Poultry science.

[9]  R. Tsien.  Fluorescent indicators of ion concentrations. , 1989, Methods in cell biology.

[10]  R. Hammerstedt,et al.  In vitro evaluation of sperm quality: an opinion. , 1993, Journal of andrology.

[11]  L. Johnson,et al.  Porcine sperm viability, oocyte fertilization and embryo development after staining spermatozoa with SYBR-14. , 1996, Theriogenology.

[12]  G. Schatten,et al.  Ubiquitin-based sperm assay for the diagnosis of male factor infertility , 2001 .

[13]  P. Williamson,et al.  Merocyanine 540, a fluorescent probe sensitive to lipid packing. , 1983, Biochimica et biophysica acta.

[14]  R. Foote,et al.  Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility. , 1998, Theriogenology.

[15]  D. Mortimer,et al.  Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon. , 1987, Journal of reproduction and fertility.

[16]  T. Davies,et al.  The plane of first cleavage is not related to the distribution of sperm components in the mouse. , 2002, Human reproduction.

[17]  Zeng-Ming Yang,et al.  Assessment of sperm viability, mitochondrial activity, capacitation and acrosome intactness in extended boar semen during long-term storage. , 2002, Theriogenology.

[18]  B. T. Storey,et al.  Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction , 1979, The Journal of cell biology.

[19]  D. Pinkel,et al.  Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses. , 1986, Biology of reproduction.

[20]  R. Ortíz,et al.  EXPRESSION OF LECTIN RECEPTORS ON THE MEMBRANE SURFACE OF SPERM OF FERTILE AND SUBFERTILE BOARS BY FLOW CYTOMETRY , 2002, Archives of andrology.

[21]  B. Ballachey,et al.  The sperm chromatin structure assay. Relationship with alternate tests of semen quality and heterospermic performance of bulls. , 1988, Journal of andrology.

[22]  N. Miller,et al.  Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: Elimination of labeling artifacts , 1999, Molecular reproduction and development.

[23]  L. Johnson,et al.  Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws. , 1988, Journal of animal science.

[24]  E. Clegg,et al.  Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. , 1999, Human reproduction.

[25]  G. Evans,et al.  Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa. , 1997, Molecular human reproduction.

[26]  C. Thomas,et al.  Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa. , 1997, Biology of reproduction.

[27]  N. Miller,et al.  Flow cytometric studies of bicarbonate‐mediated Ca2+ influx in boar sperm populations , 1993, Molecular reproduction and development.

[28]  H. Rodríguez-Martínez,et al.  Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen-thawed semen from Swedish AI bulls. , 2001, Theriogenology.

[29]  J. M. Plummer,et al.  Flow cytometric detection of bicarbonate‐induced changes in lectin binding in boar and ram sperm populations , 1995, Molecular reproduction and development.

[30]  C. Thomas,et al.  Fluorometric assessments of acrosomal integrity and viability in cryopreserved bovine spermatozoa. , 1997, Biology of reproduction.

[31]  M. Melamed,et al.  Relation of mammalian sperm chromatin heterogeneity to fertility. , 1980, Science.

[32]  G. Althouse,et al.  Assessment of boar sperm viability using a combination of two fluorophores. , 1995, Theriogenology.

[33]  A. de Kruif,et al.  Assessment of different sperm quality parameters to predict in vitro fertility of bulls. , 2002, Reproduction in domestic animals = Zuchthygiene.

[34]  M. Betancourt,et al.  ACROSOME REACTION IN FERTILE AND SUBFERTILE BOAR SPERM , 2002, Archives of andrology.

[35]  H. Rodríguez-Martínez,et al.  Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry. , 2003, Animal reproduction science.

[36]  J. Guérin,et al.  Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. , 2003, Human reproduction.

[37]  L. Johnson,et al.  Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. , 1995, Biology of reproduction.

[38]  L. M. Thurston,et al.  Identification of Amplified Restriction Fragment Length Polymorphism Markers Linked to Genes Controlling Boar Sperm Viability Following Cryopreservation1 , 2002, Biology of reproduction.

[39]  G. Evans,et al.  Capacitation status and fertility of fresh and frozen-thawed ram spermatozoa. , 1997, Reproduction, fertility, and development.

[40]  S. Hui,et al.  Merocyanine interaction with phosphatidylcholine bilayers. , 1993, Biochimica et biophysica acta.

[41]  E. Squires,et al.  Comparison of the fertility of cryopreserved stallion spermatozoa with sperm motion analyses, flow cytometric evaluation, and zona-free hamster oocyte penetration. , 1996, Theriogenology.

[42]  L. Mátyus,et al.  Flow cytometric analysis of viability of bull sperm cells. , 1984, Acta biochimica et biophysica; Academiae Scientiarum Hungaricae.

[43]  G. Sachdeva,et al.  Progesterone Receptor as an Indicator of Sperm Function , 2002, Biology of reproduction.

[44]  C. Peterson,et al.  SPERM DNA FRAGMENTATION IS INCREASED IN COUPLES WITH UNEXPLAINED RECURRENT PREGNANCY LOSS , 2003, Archives of andrology.

[45]  R. Haugland,et al.  Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. , 1994, Journal of andrology.

[46]  G. Evans,et al.  Fate and functional integrity of fresh and frozen-thawed ram spermatozoa following intrauterine insemination. , 1999, Reproduction, fertility, and development.

[47]  B. Gadella,et al.  The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane. , 2000, Development.

[48]  D. Evenson,et al.  Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility. , 1994, Theriogenology.

[49]  L. Johnson,et al.  Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation , 1997, Molecular reproduction and development.

[50]  J. Graham.  Assessment of sperm quality: a flow cytometric approach. , 2001, Animal reproduction science.

[51]  H. Rodríguez-Martínez,et al.  Relationship between the proportion of capacitated spermatozoa present in frozen-thawed bull semen and fertility with artificial insemination. , 1999, International journal of andrology.

[52]  Saima Hameed,et al.  Differential Ubiquitination of Stallion Sperm Proteins: Possible Implications for Infertility and Reproductive Seasonality1 , 2003, Biology of reproduction.

[53]  R. Harrison,et al.  Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. , 1990, Journal of reproduction and fertility.

[54]  D. Evenson,et al.  Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry. , 1990, Journal of dairy science.

[55]  B. Ball,et al.  Assessment of equine sperm mitochondrial function using JC-1. , 2000, Theriogenology.

[56]  G. Evans,et al.  The interaction of fresh and frozen-thawed ram spermatozoa with oviducal epithelial cells in vitro. , 2000, Reproduction, fertility, and development.

[57]  I. Georgiou,et al.  Chromomycin A3-staining as an indicator of protamine deficiency and fertilization. , 1996, International journal of andrology.

[58]  B. Gadella,et al.  Capacitation Induces Cyclic Adenosine 3′,5′-Monophosphate-Dependent, but Apoptosis-Unrelated, Exposure of Aminophospholipids at the Apical Head Plasma Membrane of Boar Sperm Cells1 , 2002, Biology of reproduction.

[59]  N. Miller,et al.  Bicarbonate/CO2, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membranes , 1996, Molecular reproduction and development.

[60]  G. Evans,et al.  Normal fertility in ewes after cervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma. , 1999, Reproduction, fertility, and development.

[61]  P. F. Watson,et al.  Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation. , 2001, Reproduction.

[62]  M. Nasr-Esfahani,et al.  ANDROLOGY: Relation Between Different Human Sperm Nuclear Maturity Tests and In Vitro Fertilization , 2001, Journal of Assisted Reproduction and Genetics.

[63]  C. Thomas,et al.  Effect of cryopreservation of bovine sperm organelle function and viability as determined by flow cytometry. , 1998, Biology of reproduction.

[64]  J. Overstreet,et al.  Two simple methods for detecting acrosome‐reacted human sperm , 1986 .

[65]  TsuyoshiKasai,et al.  Relationship between sperm mitochondrial membrane potential,sperm motility,and fertility potential , 2002 .