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Papers by Kathryn Robertson

Journal of andrology

Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and ... more Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and subsequent fusion with an oocyte. Sperm gain the capability to acrosome react after a period of capacitation, which primarily involves biochemical changes in the sperm membranes. The morphological events of the acrosome reaction have been well-documented, but the underlying cellular mechanisms that regulate capacitation and the acrosome reaction remain unclear. Antibodies to the 2 ubiquitous calpains, mu and m, as well as the small subunit, which associates with both calpains, were localized at the ultrastructural level to the region between the plasma membrane and the outer acrosomal membrane of cynomolgus macaque sperm. After the acrosome reaction, all of the anti-calpain antibodies labeled the acrosomal shroud, suggesting that calpains are located throughout the cytoplasmic area between the 2 outer sperm membranes. Calpastatin is an endogenous modulator of calpain activity and is also...

Journal of andrology

Subfertility in stallions is common, and methodologies are needed to increase the fertility in th... more Subfertility in stallions is common, and methodologies are needed to increase the fertility in these animals. In other species, removal of the dead sperm from semen increases the quality and fertility of semen. With horse semen we evaluated 48 combinations of column separation techniques using micro-spin chromatography columns. The greatest improvement in motility was observed with glass wool, whereas glass beads exhibited the greatest recovery of motile sperm. Although centrifugation time did not influence recovery rate or percent motility, a column length of 2 cm was superior for recovery of motile sperm. In scale-up experiments using 2-cm columns of glass beads in 50-ml syringe barrels, centrifugation proved to be superior to gravity flow, suction, and syringe plunger as extraction methods for drawing semen through the column; however, gravity flow produced acceptable results and may be more suitable for use in a field setting. When the volume of semen for separation was increase...

Fertility and sterility, 1994

To compare sperm migration through sodium hyaluronate with simple washing as methods for preparin... more To compare sperm migration through sodium hyaluronate with simple washing as methods for preparing sperm for IUI. Ten normal semen specimens were prospectively collected and samples were prepared by simple washing and by migration into sodium hyaluronate using the Sperm Select System (Select Medical Systems, Williston, VT). The semen and each treatment group were evaluated for sperm concentration, percent motile, viability, acrosomal status, longevity, and computer-aided semen analysis (CASA) parameters. University reproductive endocrinology facility. The recovery of motile sperm was significantly higher for the washing method (mean +/- SEM 75% +/- 7%) than for the hyaluronate method (10% +/- 1%). The number of motile sperm recovered by migration into hyaluronate was independent of the percentage of motile sperm in the semen specimen and positively correlated with sperm concentration. The hyaluronate method produced greater percentages of motile, viable, and morphologically normal s...

Journal of andrology

An acrosomal staining technique that can differentiate between living and dead sperm was develope... more An acrosomal staining technique that can differentiate between living and dead sperm was developed for equine sperm. The fluoresceinated lectin Pisum sativum agglutinin (FITC-PSA) was used to identify the presence or absence of acrosomal contents, while the supravital nuclear dye Hoechst 33258 (H258) was used to assess viability. The accuracy of the FITC-PSA acrosomal stain was tested by comparing the percentage of sperm that had lost their acrosomal contents, detected by the staining method, with that detected by transmission electron microscopy (TEM). Following capacitation in vitro, the acrosomal status of sperm induced to acrosome react with A23187 and of control sperm were very similar with the staining technique and TEM, confirming the accuracy of the FITC-PSA acrosomal stain. We investigated the relationship between viability as measured by exclusion of H258 and motility as measured by three methods: one subjective and two objective. Although there was a good correlation betw...

Journal of andrology

PH-20 is a glycoprotein located on the surface of the sperm plasma membrane and on the inner acro... more PH-20 is a glycoprotein located on the surface of the sperm plasma membrane and on the inner acrosomal membrane. The best understood function of sperm surface PH-20 is its hyaluronidase activity, which results in hydrolysis of the hyaluronic acid-rich cumulus matrix during sperm penetration of this extracellular oocyte investment. In this study, we investigated whether alterations in the secondary and tertiary structures of sperm surface PH-20 would affect its enzyme activity. Proteins were isolated from the sperm plasma membrane by treatment of living cells with phosphatidylinositol-specific phospholipase C (PI-PLC). PH-20 was purified from the PI-PLC released proteins by immunoaffinity chromatography. Two-dimensional electrophoresis of purified PH-20 revealed 6 isoforms with isoelectric points ranging from 5.1 to 6.0. Removal of the N-linked glycans from PH-20 with N-glycosidase F shifted the molecular weight from 64 kd to approximately 54 kd, its deduced molecular weight based on...

Reproduction, 2001

The mammalian sperm hyaluronidase, PH-20, is active in macaque spermatozoa at neutral and acid pH... more The mammalian sperm hyaluronidase, PH-20, is active in macaque spermatozoa at neutral and acid pH. Antibodies were produced to synthesized peptides representing regions of PH-20 that may be involved in hyaluronidase activity and designated peptide 1 (amino acid sequence 142-172) and peptide 3 (amino acid sequence 277-297). Western blotting of proteins extracted from the surface of acrosome-intact spermatozoa showed that the two peptide-specific, affinity-purified IgGs label a 64 kDa band corresponding to the PH-20 molecule. Western blots of acrosome-reacted spermatozoa showed that, under reducing conditions, the two anti-peptide IgGs label the 44 kDa band only, which represents the N-terminal fragment of PH-20. Anti-peptide 3 IgG also labels the 53 kDa form of PH-20 in extracts of acrosome-reacted spermatozoa. Peptide-specific, affinity-purified Fab fragments from both IgGs were shown by fluorescence microscopy and transmission electron microscopy to label the sperm plasma membrane,...

Proceedings of the National Academy of Sciences, 2004

Genetics, 2010

We have leveraged the reference sequence of a boxer to construct the first complete linkage map f... more We have leveraged the reference sequence of a boxer to construct the first complete linkage map for the domestic dog. The new map improves access to the dog's unique biology, from human disease counterparts to fascinating evolutionary adaptations. The map was constructed with ∼3000 microsatellite markers developed from the reference sequence. Familial resources afforded 450 mostly phase-known meioses for map assembly. The genotype data supported a framework map with ∼1500 loci. An additional ∼1500 markers served as map validators, contributing modestly to estimates of recombination rate but supporting the framework content. Data from ∼22,000 SNPs informing on a subset of meioses supported map integrity. The sex-averaged map extended 21 M and revealed marked region- and sex-specific differences in recombination rate. The map will enable empiric coverage estimates and multipoint linkage analysis. Knowledge of the variation in recombination rate will also inform on genomewide patte...

Animal Reproduction Science, 1997

Cryopreserved semen has been utilised in the artificial insemination of livestock species for ove... more Cryopreserved semen has been utilised in the artificial insemination of livestock species for over 40 years, even though the detrimental effects of cryopreservation on sperm function and fertility are well documented. In the present study, computer-automated sperm-head morphometry was used to determine if goat sperm-head morphometry was affected by freezing and thawing. A microscope slide was prepared from single semen samples, collected by artificial vagina, from 10 sexually active Saanen bucks. The remainder of each sample was frozen in a tris-citrate-yolk extender. After thawing, semen smears were prepared on microscope slides. All slides were stained in haematoxylin and mean sperm-head measurements of length, width, width/length, area and perimeter were determined for each slide by computer aided sperm morphometry analysis. The effects of sperm freezing on sperm-head dimensions within and among all bucks were determined. No significant (P > 0.10) freezing effect was found between fresh semen and postthaw samples for length (7.00 microns vs 7.13 microns), width (3.77 microns vs 3.87 microns), width/length (0.54 micron vs 0.54 micron), area (19.67 microns2 vs 20.57 microns2) and perimeter (18.62 microns vs 18.83 microns) when analysed across all bucks. Significant differences (P < 0.05) were however found within three bucks for area, perimeter, length and width, with the percentage increase in measurements being significantly greater than in the remaining bucks. The variability of the morphometric dimensions were not affected by freezing. The results indicate that semen freezing did not affect the overall dimensions of sperm heads across the entire population of bucks sampled. However, since sperm-head dimensions from three bucks were affected, changes in sperm-head morphometry may be indicative of spermatozoa of the semen from individuals to successfully freeze. Because the overall mean sperm-head dimensions acquired from frozen/thawed semen were not different from those of fresh semen, previously reported measurements of goat sperm heads are probably reflective of fresh semen. More importantly, retrospective studies of sperm-head morphometry and fertility may now be performed utilising extensive breeding records from frozen semen.

Animal Genetics, 2013

Although variation in the KIT gene is a common cause of white spotting among domesticated animals... more Although variation in the KIT gene is a common cause of white spotting among domesticated animals, KIT has not been implicated in the diverse white spotting observed in the dog. Here, we show that a loss-of-function mutation in KIT recapitulates the coat color phenotypes observed in other species. A spontaneous white spotting observed in a pedigree of German Shepherd dogs was mapped by linkage analysis to a single locus on CFA13 containing KIT (pairwise LOD = 15). DNA sequence analysis identified a novel 1-bp insertion in the second exon that co-segregated with the phenotype. The expected frameshift and resulting premature stop codons predicted a severely truncated c-Kit receptor with presumably abolished activity. No dogs homozygous for the mutation were recovered from multiple intercrosses (P = 0.01), suggesting the mutation is recessively embryonic lethal. These observations are consistent with the effects of null alleles of KIT in other species.

Animal Biotechnology, 2002

Animal Biotechnology, 2012

Journal of andrology

Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and ... more Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and subsequent fusion with an oocyte. Sperm gain the capability to acrosome react after a period of capacitation, which primarily involves biochemical changes in the sperm membranes. The morphological events of the acrosome reaction have been well-documented, but the underlying cellular mechanisms that regulate capacitation and the acrosome reaction remain unclear. Antibodies to the 2 ubiquitous calpains, mu and m, as well as the small subunit, which associates with both calpains, were localized at the ultrastructural level to the region between the plasma membrane and the outer acrosomal membrane of cynomolgus macaque sperm. After the acrosome reaction, all of the anti-calpain antibodies labeled the acrosomal shroud, suggesting that calpains are located throughout the cytoplasmic area between the 2 outer sperm membranes. Calpastatin is an endogenous modulator of calpain activity and is also...

Journal of andrology

Subfertility in stallions is common, and methodologies are needed to increase the fertility in th... more Subfertility in stallions is common, and methodologies are needed to increase the fertility in these animals. In other species, removal of the dead sperm from semen increases the quality and fertility of semen. With horse semen we evaluated 48 combinations of column separation techniques using micro-spin chromatography columns. The greatest improvement in motility was observed with glass wool, whereas glass beads exhibited the greatest recovery of motile sperm. Although centrifugation time did not influence recovery rate or percent motility, a column length of 2 cm was superior for recovery of motile sperm. In scale-up experiments using 2-cm columns of glass beads in 50-ml syringe barrels, centrifugation proved to be superior to gravity flow, suction, and syringe plunger as extraction methods for drawing semen through the column; however, gravity flow produced acceptable results and may be more suitable for use in a field setting. When the volume of semen for separation was increase...

Fertility and sterility, 1994

To compare sperm migration through sodium hyaluronate with simple washing as methods for preparin... more To compare sperm migration through sodium hyaluronate with simple washing as methods for preparing sperm for IUI. Ten normal semen specimens were prospectively collected and samples were prepared by simple washing and by migration into sodium hyaluronate using the Sperm Select System (Select Medical Systems, Williston, VT). The semen and each treatment group were evaluated for sperm concentration, percent motile, viability, acrosomal status, longevity, and computer-aided semen analysis (CASA) parameters. University reproductive endocrinology facility. The recovery of motile sperm was significantly higher for the washing method (mean +/- SEM 75% +/- 7%) than for the hyaluronate method (10% +/- 1%). The number of motile sperm recovered by migration into hyaluronate was independent of the percentage of motile sperm in the semen specimen and positively correlated with sperm concentration. The hyaluronate method produced greater percentages of motile, viable, and morphologically normal s...

Journal of andrology

An acrosomal staining technique that can differentiate between living and dead sperm was develope... more An acrosomal staining technique that can differentiate between living and dead sperm was developed for equine sperm. The fluoresceinated lectin Pisum sativum agglutinin (FITC-PSA) was used to identify the presence or absence of acrosomal contents, while the supravital nuclear dye Hoechst 33258 (H258) was used to assess viability. The accuracy of the FITC-PSA acrosomal stain was tested by comparing the percentage of sperm that had lost their acrosomal contents, detected by the staining method, with that detected by transmission electron microscopy (TEM). Following capacitation in vitro, the acrosomal status of sperm induced to acrosome react with A23187 and of control sperm were very similar with the staining technique and TEM, confirming the accuracy of the FITC-PSA acrosomal stain. We investigated the relationship between viability as measured by exclusion of H258 and motility as measured by three methods: one subjective and two objective. Although there was a good correlation betw...

Journal of andrology

PH-20 is a glycoprotein located on the surface of the sperm plasma membrane and on the inner acro... more PH-20 is a glycoprotein located on the surface of the sperm plasma membrane and on the inner acrosomal membrane. The best understood function of sperm surface PH-20 is its hyaluronidase activity, which results in hydrolysis of the hyaluronic acid-rich cumulus matrix during sperm penetration of this extracellular oocyte investment. In this study, we investigated whether alterations in the secondary and tertiary structures of sperm surface PH-20 would affect its enzyme activity. Proteins were isolated from the sperm plasma membrane by treatment of living cells with phosphatidylinositol-specific phospholipase C (PI-PLC). PH-20 was purified from the PI-PLC released proteins by immunoaffinity chromatography. Two-dimensional electrophoresis of purified PH-20 revealed 6 isoforms with isoelectric points ranging from 5.1 to 6.0. Removal of the N-linked glycans from PH-20 with N-glycosidase F shifted the molecular weight from 64 kd to approximately 54 kd, its deduced molecular weight based on...

Reproduction, 2001

The mammalian sperm hyaluronidase, PH-20, is active in macaque spermatozoa at neutral and acid pH... more The mammalian sperm hyaluronidase, PH-20, is active in macaque spermatozoa at neutral and acid pH. Antibodies were produced to synthesized peptides representing regions of PH-20 that may be involved in hyaluronidase activity and designated peptide 1 (amino acid sequence 142-172) and peptide 3 (amino acid sequence 277-297). Western blotting of proteins extracted from the surface of acrosome-intact spermatozoa showed that the two peptide-specific, affinity-purified IgGs label a 64 kDa band corresponding to the PH-20 molecule. Western blots of acrosome-reacted spermatozoa showed that, under reducing conditions, the two anti-peptide IgGs label the 44 kDa band only, which represents the N-terminal fragment of PH-20. Anti-peptide 3 IgG also labels the 53 kDa form of PH-20 in extracts of acrosome-reacted spermatozoa. Peptide-specific, affinity-purified Fab fragments from both IgGs were shown by fluorescence microscopy and transmission electron microscopy to label the sperm plasma membrane,...

Proceedings of the National Academy of Sciences, 2004

Genetics, 2010

We have leveraged the reference sequence of a boxer to construct the first complete linkage map f... more We have leveraged the reference sequence of a boxer to construct the first complete linkage map for the domestic dog. The new map improves access to the dog's unique biology, from human disease counterparts to fascinating evolutionary adaptations. The map was constructed with ∼3000 microsatellite markers developed from the reference sequence. Familial resources afforded 450 mostly phase-known meioses for map assembly. The genotype data supported a framework map with ∼1500 loci. An additional ∼1500 markers served as map validators, contributing modestly to estimates of recombination rate but supporting the framework content. Data from ∼22,000 SNPs informing on a subset of meioses supported map integrity. The sex-averaged map extended 21 M and revealed marked region- and sex-specific differences in recombination rate. The map will enable empiric coverage estimates and multipoint linkage analysis. Knowledge of the variation in recombination rate will also inform on genomewide patte...

Animal Reproduction Science, 1997

Cryopreserved semen has been utilised in the artificial insemination of livestock species for ove... more Cryopreserved semen has been utilised in the artificial insemination of livestock species for over 40 years, even though the detrimental effects of cryopreservation on sperm function and fertility are well documented. In the present study, computer-automated sperm-head morphometry was used to determine if goat sperm-head morphometry was affected by freezing and thawing. A microscope slide was prepared from single semen samples, collected by artificial vagina, from 10 sexually active Saanen bucks. The remainder of each sample was frozen in a tris-citrate-yolk extender. After thawing, semen smears were prepared on microscope slides. All slides were stained in haematoxylin and mean sperm-head measurements of length, width, width/length, area and perimeter were determined for each slide by computer aided sperm morphometry analysis. The effects of sperm freezing on sperm-head dimensions within and among all bucks were determined. No significant (P > 0.10) freezing effect was found between fresh semen and postthaw samples for length (7.00 microns vs 7.13 microns), width (3.77 microns vs 3.87 microns), width/length (0.54 micron vs 0.54 micron), area (19.67 microns2 vs 20.57 microns2) and perimeter (18.62 microns vs 18.83 microns) when analysed across all bucks. Significant differences (P < 0.05) were however found within three bucks for area, perimeter, length and width, with the percentage increase in measurements being significantly greater than in the remaining bucks. The variability of the morphometric dimensions were not affected by freezing. The results indicate that semen freezing did not affect the overall dimensions of sperm heads across the entire population of bucks sampled. However, since sperm-head dimensions from three bucks were affected, changes in sperm-head morphometry may be indicative of spermatozoa of the semen from individuals to successfully freeze. Because the overall mean sperm-head dimensions acquired from frozen/thawed semen were not different from those of fresh semen, previously reported measurements of goat sperm heads are probably reflective of fresh semen. More importantly, retrospective studies of sperm-head morphometry and fertility may now be performed utilising extensive breeding records from frozen semen.

Animal Genetics, 2013

Although variation in the KIT gene is a common cause of white spotting among domesticated animals... more Although variation in the KIT gene is a common cause of white spotting among domesticated animals, KIT has not been implicated in the diverse white spotting observed in the dog. Here, we show that a loss-of-function mutation in KIT recapitulates the coat color phenotypes observed in other species. A spontaneous white spotting observed in a pedigree of German Shepherd dogs was mapped by linkage analysis to a single locus on CFA13 containing KIT (pairwise LOD = 15). DNA sequence analysis identified a novel 1-bp insertion in the second exon that co-segregated with the phenotype. The expected frameshift and resulting premature stop codons predicted a severely truncated c-Kit receptor with presumably abolished activity. No dogs homozygous for the mutation were recovered from multiple intercrosses (P = 0.01), suggesting the mutation is recessively embryonic lethal. These observations are consistent with the effects of null alleles of KIT in other species.

Animal Biotechnology, 2002

Animal Biotechnology, 2012