A novel nozzle for more efficient sperm orientation to improve sorting efficiency of X and Y chromosome-bearing sperm

Rens, W.; Welch, G. Rickey; Johnson, Lawrence A.

doi:10.1002/(sici)1097-0320(19981201)33:4<476::aid-cyto12>3.0.co;2-b

Cited by 60 publications

(37 citation statements)

References 19 publications

(6 reference statements)

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“…Isolations were performed using a flow cytometer with specific modifications as developed by Johnson et al (13). Modifications included an additional fluorescence detector positioned at a right angle (90 o ) and a novel sample injection tip designed to "orient" the spermatozoa to the laser beam (14,15). Fluorescent signal from Hoechst-stained spermatozoa oriented with their brightest edge toward the 90 o detector was collected by 0 o detector and showed bimodal distribution.…”

Section: Resultsmentioning

confidence: 99%

CAG Repeat Lengths in X- and Y-bearing Sperm Indicate That Gender Bias during Transmission of Huntington's Disease Gene Is Determined in the Embryo

Kovtun

Welch

Guthrie

et al. 2004

Journal of Biological Chemistry

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The size of the CAG tract at the Huntington's disease (HD) locus upon transmission depends on the gender of the parent. However, the basis for the parent-of-origin effect is unknown. To test whether expansion and contraction in HD are "imprinted" in the germ cells, we isolated the X-and Y-bearing sperm of HD transgenic mice. Here we show that CAG repeat distributions in the X-and Y-bearing spermatozoa of founding fathers do not differ. These data show that gender-dependent changes in CAG repeat length arise in the embryo.The mechanism for expansion in HD 1 and other trinucleotide disorders is not known. However, one of the poorly understood features of expansion is the gender bias that is associated with transmission. Large changes in repeat number are known to be transmitted through the paternal line in HD (1, 2), SCA1 (3), dentatorubral-pallidoluysian atrophy (DRPLA) (4), MachadoJoseph disease (5), and spinal and bulbar muscular atrophy (SBMA) (6) and through the maternal line in Fragile X (7, 8). The gender bias transmission has also been documented in transgenic mice (9 -12). Similar to humans, HD transgenic mice transmit expansions predominantly through the male germ line (9,10,12). Moreover, we found that expansion and contraction of CAG repeat size in the hHD transgene depended on the gender of the progeny (9).Expansions are primarily seen in males and contractions dominate in females. The molecular basis for the parent-oforigin effect is unknown. The gender dependence of expansion can be due to differences in CAG repeat distribution in X-and Y-bearing sperm. Alternatively, the change in repeat number, contraction, or expansion can take place after fertilization in early embryogenesis. In order to distinguish between these two mechanisms, the isolation and purification of the X-and Ybearing sperm were required. This, however, was not possible using conventional technology.In present work, we have developed techniques and used advanced technology to separate pure populations of X-and Y-bearing germ cells from HD transgenic mice. We show here that CAG repeat distributions in X-and Y-bearing parental sperm are not different. Repeat expansion occurs equally well within both X and Y chromosomes. This indicates that the gender-dependent processing of CAG repeats must take place post-zygotically. Thus, these data provide evidence for a new kind of imprinting that may be important in the interpretation of genetic data in other systems. EXPERIMENTAL PROCEDURESAnimals-HD transgenic male mice (line B6CBA-TgN R6/1) were originally purchased from The Jackson Laboratory. The colony was maintained at the animal core facility, Mayo Clinic/Foundation. Animals were routinely screened for the presence of HD transgene by PCR (10, 18).Preparation of Single Cell Suspension and Flow Sorting-Mouse epididymis was dissected from the animal, placed in phosphate-buffered saline, and chopped with a razor. Resulting suspension was filtered through cheesecloth. The supernatant was centrifuged at 1500 rpm, and the pellet was resuspended...

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Section: Resultsmentioning

confidence: 99%

CAG Repeat Lengths in X- and Y-bearing Sperm Indicate That Gender Bias during Transmission of Huntington's Disease Gene Is Determined in the Embryo

Kovtun

Welch

Guthrie

et al. 2004

Journal of Biological Chemistry

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“…10 6 spermatozoa/ml with staining medium and incubated with H33342 (Sigma-Aldrich; 89-107 mM) as previously described (O'Brien & Robeck 2006). A high-speed flow cytometer (SX MoFlo, Dako Colorado, Inc., Fort Collins, CO, USA) modified for sperm sorting (Rens et al 1998, Sharpe & Evans 2009) operated at 207 kPA (30 psi) was used to analyze and sort spermatozoa. Spermatozoa were sorted into 3 ml of Part A (with homologous 1% seminal plasma, v/v, prepared as previously described; O'Brien & Robeck 2006) for a maximum of 2 h, and then centrifuged at 850 g for 25 min at room temperature.…”

Section: Sorting Of Frozen-thawed Spermatozoamentioning

confidence: 99%

Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

Montano¹,

Kraemer²,

Love³

et al. 2012

Reproduction

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Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P!0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P!0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6G4.1%) at 6 h after the second thawing step and remained unchanged (PO0.05) at 24 h. The viability of sorted spermatozoa was higher (P!0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

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“…Major improvements, including the use of a bevelled needle and an orientation nozzle tip with double elliptic torsions that promote the alignment of spermatozoa in front of the laser, were developed (Rens et al 1998). These modifications resulted in a fourfold increase in the number of sorted spermatozoa produced per unit of time.…”

Section: Sex Sorting Of Spermatozoa By Flow Cytometrymentioning

confidence: 99%

“…Inserted into a high-speed flow cytometer, sort rates of 12-15 million spermatozoa per hour became a reality and were the prerequisite for commercial application . While this 'HISON' orientating nozzle (Rens et al 1998) had a double torsional elliptic shape, most commercial sorters today work with a further refined single torsion nozzle (Cytonozzle; XY, Inc., Fort Collins, CO, USA) for better sperm orientation. Recently, we developed an updated version of the double torsional nozzle with an improved internal R16 D Rath and others geometry in order to optimise the efficiency of sperm orientation.…”

Section: Sex Sorting Of Spermatozoa By Flow Cytometrymentioning

confidence: 99%

Sex selection of sperm in farm animals: status report and developmental prospects

Rath¹,

Barcikowski²,

Graaf³

et al. 2013

Reproduction

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Pre-selection of spermatozoa based on the relative DNA difference between X-and Y-chromosome bearing populations by flow cytometry is an established method that has been introduced into commercial cattle production. Although several important improvements have increased the sort efficiency, the fertilising ability of sexed spermatozoa based on offspring per insemination is still behind farmers' expectations. The main stress factors, especially on mitochondria, that reduce the lifespan of spermatozoa are described, and new technical as well as biological solutions to maintain the natural sperm integrity and to increase the sorting efficiency are discussed. Among these methods are the identification of Y-chromosome bearing spermatozoa by bi-functionalised gold nanoparticles and triplex hybridisation in vivo as well as new laser-controlled deflection system that replaces the deflection of spermatozoa in the electrostatic field. Additionally, as well as a new nonsurgical transfer system of spermatozoa into the oviduct of cows has been developed and allows a significant reduction of spermatozoa per transfer. Altogether, the improvements made in the recent years will allow a broader use of sex-sorted spermatozoa even in those species that require more cells than cows and sheep.Reproduction (2013) 145 R15-R30

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A novel nozzle for more efficient sperm orientation to improve sorting efficiency of X and Y chromosome-bearing sperm

Cited by 60 publications

References 19 publications

CAG Repeat Lengths in X- and Y-bearing Sperm Indicate That Gender Bias during Transmission of Huntington's Disease Gene Is Determined in the Embryo

CAG Repeat Lengths in X- and Y-bearing Sperm Indicate That Gender Bias during Transmission of Huntington's Disease Gene Is Determined in the Embryo

Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

Sex selection of sperm in farm animals: status report and developmental prospects

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