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Biomedical subjects

D J McLean

Publications and source records attributed to D J McLean.

17 recordsLinked to original sources
PubMed2005

Changes in spermatogenesis and endocrine function in the ram testis due to irradiation and active immunization against luteinizing hormone-releasing hormone.

Spermatogonial stem cell transplantation is a technique that has potential in livestock to enhance genetic gain and generate transgenic offspring through the male germ line. A means for depletion of endogenous germ cells in a recipient's seminiferous tubules is necessary for this technology to be applied. The objectives of this study were to evaluate several methods for depletion of endogenous germ cells in the testes of adult rams and to evaluate ultrasound-guided injections into the rete testes as a means for infusing a suspension into the seminiferous tubules. Sixteen adult rams were randomly divided into 4 treatment groups (n = 4 per group). Treatments consisted of active immunization against LHRH (IMM), localized testicular irradiation (IR), LHRH immunization + irradiation (IMM+IR), and untreated control. Serial bleedings were conducted pretreatment and monthly after treatment for 4 mo, at which time all rams were castrated. Both IMM and IMM+IR rams received exogenous gonadotropin in the form of Perganol weekly for 8 wk before castration to bypass the immunization. All rams also received an ultrasound-guided injection of PBS containing 0.4% trypan blue into the rete testis of one testicle before castration. Rams receiving IMM and IMM+IR treatments had higher (P < 0.05) average percentages of seminiferous tubule cross sections with depleted germ cells compared with controls. Serum testosterone was decreased (P < 0.05) in IMM and IMM+IR rams 1 mo after treatment and throughout the remainder of the study compared with controls and IR rams, which were not different from each other. Serum inhibin concentration was unchanged in all rams following treatment indicating that Sertoli cell function was unaltered. A greater (P < 0.05) average percentage of the total testicular area could be filled with the trypan blue solution by rete testis injection in IMM and IMM+IR rams. These data demonstrate the depletion of endogenous germ cells in adult ram testes without alteration of Sertoli cell viability and function that have potential as methods for preparing recipient animals for germ cell transplantation.

Animals↗
PubMed2002

Transplantation of bovine germinal cells into mouse testes.

To develop techniques for spermatogonial transplantation in bulls, it is essential to have an effective bioassay procedure to evaluate the transplantation efficiency of spermatogonial stem cell collection, purification, and culture techniques. The objective of the present study was to develop a mouse bioassay model to evaluate transplantation efficiency of fresh and cultured bovine germ cells. Bull calves of four ages (1, 2, 3, and 4 mo) were used as a source of donor testes cells. Two calves were used for each age point, one calf was experimentally made cryptorchidistic at 1 wk of age and the other left normal. A STO (mouse fibroblast) feeder cell line was used to culture bovine testes cells for 2 wk preceding transfer into recipient testes. Immunodeficient nude mice (nu/nu) in which endogenous spermatogenesis had been abolished by busulfan treatment served as recipient animals for transplantation. Donor bovine germ cells were microinjected into mouse seminiferous tubules. Mouse testes were analyzed 2 wk after transplant with the use of a bovine-specific antibody and whole-mount immunohistochemistry for the presence of bovine donor germ cells. Bovine testis cells were present in all recipient mouse testes analyzed. Fresh bovine testes cells were observed as colonies of round cells within mouse seminiferous tubules, indicating spermatogonial expansion and colonization; however, cultured bovine testes cells appeared as fibrous tissue and not as spermatogenic colonies. The average number of colonies resulting from donor cryptorchid testes was not different (P > 0.05) from noncryptorchid, 56+/-4 and 78+/-7, respectively. Fresh donor cells from calves older than 1 mo gave rise to a greater average number of colonies within recipient testes (P <0.05) (1 mo, 33+/-4; 2 mo, 70+/-8; 3 mo, 63+/-6; 4 mo, 87+/-9). Fresh bovine germ cells are capable of colonization in the busulfan-treated nude mouse testis, making it a suitable model for evaluation and development of spermatogonial transplant techniques in bulls.

Animals↗
PubMed2001

Methylation of CpG dinucleotides alters binding and silences testis-specific transcription directed by the mouse lactate dehydrogenase C promoter.

The mouse lactate dehydrogenase c gene (mldhc) is transcribed only in cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-base pair fragment was able to drive testis-specific transcription in vitro and in transgenic mice. Several testis-specific genes are believed to be regulated at least in part through differential methylation of CpG dinucleotides. We investigated the possibility that transcriptional repression of the mldhc gene is mediated in somatic tissues by hypermethylation of CpG dinucleotides. The CpG dinucleotides within a fragment of the mldhc promoter containing a GC box and tandem activating transcription factor/cAMP-responsive element binding sites are hypermethylated in somatic tissues and hypomethylated in testis. Methylation of the activating transcription factor/cAMP-responsive elements altered the protein binding pattern observed in electrophoretic mobility shift assays using mouse liver but not testis nuclear extract. Furthermore, methylation of an extended mldhc promoter fragment driving lac Z silenced transcription from the promoter in a transient transfection assay. These data suggest that tissue-specific differential methylation plays a role in mldhc silencing in somatic tissues.

Activating Transcription Factors↗
PubMed2000

E-MAP-115, encoding a microtubule-associated protein, is a retinoic acid-inducible gene required for spermatogenesis.

Cell type-specific microtubules, such as the Sertoli cell microtubules and the manchette and flagellum microtubules of the spermatids, play essential roles in spermatogenesis. We identified the gene encoding E-MAP-115 (epithelial microtubule-associated protein of 115 kD) as a retinoic acid-inducible gene using gene trap mutagenesis in mouse embryonic stem cells. The gene trap insertion led to a null allele of the E-MAP-115 gene and, in agreement with its high expression in the testis, male mice homozygous for the mutation were sterile because of deformation of spermatid nuclei and subsequent gradual loss of germ cells. Consistent with a possible role for E-MAP-115 in stabilizing microtubules, microtubule associations in the mutant were morphologically abnormal in the manchette of spermatids and in Sertoli cells. We hypothesize that the abnormal microtubules in these two cell types are responsible for deformation of spermatid nuclei and germ cell loss, respectively, and indicate an essential role for E-MAP-115 in microtubule functions required for spermatogenesis.

Animals↗
PubMed1998

Transfer of sperm into a chemically defined environment by centrifugation through 12% (wt/vol) Accudenz.

Centrifugation is commonly used to wash sperm; however, most washing techniques do not put sperm in a chemically defined environment. Rather, washing by centrifugation, in effect, dilutes seminal plasma components. A 0.5-mL volume of 30% (wt/vol) Accudenz was layered beneath 5 mL of 12% (wt/vol) Accudenz in a 15-mL polypropylene centrifuge tube. Diluted semen from individual males (n = 10) was overlaid upon the 12% (wt/vol) Accudenz. After centrifugation at 1,250 x g at 4 C for 25 min, washed sperm were present at the interface of the Accudenz layers. Based upon hemacytometer counts, sperm recovery was 83% (CV = 12%). Neither sperm viability nor morphology was affected by washing. Efficacy of the washing procedure was evaluated by using extracellular glucose, glutamic acid, Ca+2, and protein as markers. Washing eliminated 99% of the glutamic acid and glucose associated with sperm. Likewise, washing removed 98.5% of the extracellular Ca+2 associated with sperm. As evidenced by total protein analysis and SDS-PAGE, washing removed 98% of soluble seminal plasma proteins from sperm. In addition, washing did not affect sperm mobility or fertilizing ability. This procedure returns extended sperm to a physiological concentration in a chemically defined environment. By suspending washed sperm in distinct media, we induced differential sperm mobility. Therefore, this procedure is suitable for the study of the effect of specific substances upon sperm cell function.

Analysis of Variance↗
PubMed1997

Increased fecundity resulting from semen donor selection based upon in vitro sperm motility.

Semen donors were selected from a population of 100 roosters based upon the extent to which sperm penetrated 6% (wt/vol) Accudenz from an overlay of extended semen. Semen donors categorized by average or high sperm motility (n = 5 per phenotype) were ejaculated weekly, their ejaculates pooled by phenotype, and pooled semen extended. A subsample of each sperm suspension was overlaid on 6% (wt/vol) Accudenz in a cuvette, the cuvette was placed in a 41 C water bath, and the absorbance of the Accudenz layer was measured after a 5-min incubation. The remainder of the sperm suspension was inseminated (n = 55 hens per phenotype). Each hen was inseminated weekly with 50 x 10(6) sperm for 14 wk. The hatchability of eggs laid by hens inseminated with sperm from the high motility phenotype was 10% greater (P < or = 0.001) than that of hens inseminated with sperm from the average phenotype. The difference in fecundity was explicable in terms of fertility (P < or = 0.001). A replicate experiment tested the effect of sperm motility as well as insemination dose on fertility. Roosters were treated as above, and hens (n = 41 to 45 per phenotype) were inseminated weekly with 25, 50, or 100 x 10(6) sperm per hen for 3 wk. Two-way ANOVA detected a sperm motility effect (P < or = 0.0001) but did not detect a dose effect (P > or = 0.05) or a motility by dose interaction (P > or = 0.05). A posteriori comparison among means revealed that the maximal fertility obtained with sperm from average roosters was 9% less (P < or = 0.05) than that obtained with only 25% as many sperm from the high motility phenotype. These experiments demonstrated that the fecundity of artificially inseminated hens can be increased when sperm penetration of Accudenz is used as a selection criterion for semen donors.

Analysis of Variance↗
PubMed1997

Reduced glucose transport in sperm from roosters (Gallus domesticus) with heritable subfertility.

Roosters homozygous for the rose comb allele (R/R) are subfertile. In previous research, these subfertile roosters were characterized by an in vitro sperm penetration assay as having limited sperm motility. The objectives in the present study were to characterize sperm motility by computer-assisted sperm motion analysis and to account for a mechanism underlying poor sperm motility. Percentages of motile sperm differed between subfertile males and fertile controls (r/r) by 29% (p < 0.001). The concentration of intracellular ATP in sperm form subfertile roosters was less than in that from fertile controls (p < 0.001). The genotypic difference is sperm motility, as measured with the sperm penetration assay, was maintained when ATP production was dependent on anaerobic glycolysis (p < 0.001). In this case, sperm were incubated with exogenous glucose and cyanide. Consequently, we could not attribute the genotypic difference in sperm mobility to mitochondrial respiration. In contrast, glucose transport, as measured by the uptake of [1,2-3H]-2-deoxy-D-glucose, was reduced in sperm from subfertile roosters (p < 0.001). Neither hexokinase nor glyceraldehyde-3-phosphate dehydrogenase activity differed between genotypes (p > 0.05). Likewise, lactate dehydrogenase activity did not differ between genotypes (p > 0.05). As evidenced by creatine kinase activity and dynein ATPase activity, neither the potential for energy transfer nor utilization within the axoneme differed between genotypes (p > 0.05). Therefore, we attribute the subfertility of roosters homozygous for the rose comb allele to decreased spermatozoal glucose transport.

Adenosine Triphosphate↗
PubMed1996

Identification of a sperm cell attribute responsible for subfertility of roosters homozygous for the rose comb allele.

Fertility, sperm metabolism, sperm filling of oviductal sperm storage tubules (SST), and sperm motility were compared in subfertile roosters homozygous for the rose comb allele (R/R) and fertile controls (r/r or R/r males). As expected, fertility of R/R males was less than that of controls (p < 0.0001). The metabolic rate of spermatozoa from R/R males was also lower than that of controls (p < 0.05). Likewise, filling of SST in vivo was lower (p < 0.0001) for spermatozoa from R/R males than for control spermatozoa. However, in vitro filling of SST was not different between genotypes (p > 0.05). Motility of spermatozoa from R/R males was less than that of controls (p < 0.001) as determined by an objective spectrophotometric assay. Previous researchers have concluded that subfertility associated with homozygosity for the rose comb allele is attributable to a sperm-specific phenomenon. However, the cellular mechanism(s) responsible for the subfertile status were not defined. In contrast to previous researchers, we have demonstrated that subfertility of R/R males is explicable in terms of reduced sperm transport through the hen's vagina; reduced sperm motility appears to be the major contributing factor.

Alleles↗
PubMed1996

Objective measurement of sperm motility based upon sperm penetration of Accudenz.

When a suspension of rooster sperm was overlaid upon 6% (wt/vol) Accudenz, immotile sperm did not enter but motile sperm entered rapidly. The absorbance of the Accudenz layer increased as a result. These phenomena were used to measure sperm motility objectively at body temperature. The intra-assay coefficient of variation (CV) was 2.6% (n = 3). When roosters (n = 36) were ejaculated repeatedly and sperm motility data analyzed by two-way ANOVA, a male effect was observed (P < or = 0.001). When roosters were ranked by mean motility scores (n = 3 evaluations per male) and representative males selected as semen donors, a difference in fertility (P < or = 0.001) was observed between males characterized by minimal and maximal sperm motility. Frequency analysis with data from a second flock of roosters (n = 100) revealed a normal distribution. Roosters categorized by average sperm motility (n = 18) or sperm motility greater than one standard deviation above the mean (n = 17) were selected for further analysis by repeated measurements. A split-plot ANOVA revealed a difference between categories (P < or = 0.0001) and variation among males within a category (P < or = 0.0001). In contrast, sperm motility was independent of time and there was no interaction between category and time. Thereafter, five roosters from each group were ejaculated weekly and interassay CV estimated with semen pooled by category (n = 3 observations per category). During this interval, sperm motility of average roosters was 55 +/- 5.9% of that of roosters within the high motility category. Interassay CV were 18.1 and 9.2% for roosters originally categorized by average and high sperm motility, respectively. The assay described has potential for: 1) selecting males based on sperm motility, and 2) standardizing the measurement of poultry sperm motility.

Analysis of Variance↗
PubMedDate not supplied

Germ cell transplantation and the study of testicular function.

Spermatogonial stem cell transplantation is a novel technique in which donor testicular cells are transferred into recipient testes. A population of germ cells from a transgenic or mutant donor is introduced into the seminiferous tubules of recipient testes by microinjection. Following injections, spermatogonial stem cells can colonize the recipient testis, initiate spermatogenesis and produce sperm capable of fertilization. This technique will allow scientists to: (1) investigate fundamental aspects of spermatogenesis; (2) provide a method to regenerate spermatogenesis in infertile individuals; and (3) genetically manipulate spermatogonial stem cells to develop transgenic animals.

Animals↗
PubMedDate not supplied

Information requirements for HMOs: the planning function.

With estimates for 1993 indicating that HMO enrollment will reach 50 million nationwide, it is apparent that HMOs have successfully overcome the obstacles they faced to gain acceptance as a valid part of the healthcare industry. However, the most crucial challenge is still being faced--effective information management.

Ambulatory Care Information Systems↗
PubMedDate not supplied

MedCenters Health Plan case study.

This case history will describe how the planning department at MedCenters Health Plan, a large multidisciplinary group practice in Minneapolis, Minn, uses its information system to support decision making.

Decision Support Systems, Management↗
PubMedDate not supplied

Isolation and characterization of mitochondria from turkey spermatozoa.

A procedure was developed to rapidly isolate functional, intact mitochondria from turkey spermatozoa. Semen was collected from turkeys, pooled, and centrifuged to remove spermiophages and other cells. The sperm cells were then mechanically disrupted with a Dounce homogenizer, sonicated, and centrifuged using a discontinuous Percoll gradient. Electron microscopy revealed morphologically intact mitochondria. The isolated mitochondria exhibited cytochrome oxidase activity, oxygen consumption, and were stained by rhodamine 123, a fluorescent stain specific for functional mitochondria in eukaryotic cells. Mitochondrial DNA (mtDNA) was isolated and purified, and the genome was determined to be 16.457 +/- 0.07 kbp. Restriction fragment patterns were identified using the endonucleases EcoR1, HindIII, and BamH1. Mitochondrial DNA was also purified from turkey liver and testis, and no differences in the restriction enzyme patterns were found between somatic and germ cell mtDNA. It is concluded that mitochondria can be isolated from spermatozoa for metabolic or genetic study.

Animals↗