270 PROGRESSIVE INCORPORATION OF CENEXIN IS RELATED TO SPERM MATURATION DURING EPIDIDYMAL TRANSIT IN THE DOMESTIC CAT

2015 ◽  
Vol 27 (1) ◽  
pp. 224
Author(s):  
T. Rowlison ◽  
M. A. Ottinger ◽  
P. Comizzoli
Keyword(s):  
Repeated Measures ◽  
Vas Deferens ◽  
Rete Testis ◽  
Domestic Cat ◽  
Sperm Maturation ◽  
Developmental Potential ◽  
Progressive Movement ◽  
Testicular Spermatozoa ◽  
Labelling Intensity ◽  
Adult Cat

The sperm centrosome is an essential organelle playing a key role just after penetration into the oocyte. It serves to organise the sperm aster, which is required for syngamy and formation of the first mitotic spindle. It is also associated with acquisition of motility during epididymal transit. Previously, we demonstrated that testicular spermatozoa exhibit reduced developmental potential after oocyte injection due to the presence of an immature centrosome [Comizzoli et al. 2006 Biol. Reprod. 75, 252–260]. Centrosome and flagellum maturation naturally occur during epididymal transit where secreted proteins impart changes on the sperm to acquire its functional properties. The objective of this study was to better understand centrosome and flagellum maturation and identify key proteins that could be used to artificially mature testicular spermatozoa. Specifically, we focused our effort on cenexin, a protein that has been reported to aid in maturation of the flagellum and somatic cell centrosome. Epididymides were dissected from adult cat testes (>1 yr old). Spermatozoa were then extracted from the different regions (caput, corpus, cauda, and vas deferens) by slicing with a scalpel blade in phosphate buffered saline at 37°C and processed separately. Control samples were also collected from the rete testis. After recording sperm motility and forward progressive movement (FPM, from 0 = immotile to 5 = fast and straight), cells were fixed in 4% paraformaldehyde and immunostained with anti-cenexin antibodies labelled with a fluorescent probe. The proportion of cells with cenexin at the location of the centrosome and the intensity of immunofluorescence were quantified (n = 8 and 4 testes, respectively). The same methods were followed for detection of cenexin in the tail portion (n = 4 testes). Statistical analyses were conducted using repeated-measures and treatments were further compared using either a protected Tukey's or F-test for orthogonal contrasts. The proportion of sperm with cenexin localised at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis and highest proportion in the cauda (45 v. 81%, T28 = 4.65, P < 0.0001). Among the labelled sperm, the intensity of immunofluorescence also significantly increased from the testis to vas deferens (4.33 v. 8.57 mean grey value; T12 = 3.29, P < 0.0065). Both motility and FPM increased from the testis to cauda segment (0 v. 93%, F4,15 = 13.53, P < 0.0001 and 0 v. 3.8 FPM, F4,15 = 26.67, P < 0.0001); however, the proportion with cenexin in the tail (range, 20 to 36%) as well as the labelling intensity (range, 3.14 to 5.26 mean grey value) did not change (P > 0.05) along the tract. These results clearly indicate that cenexin may be associated with centrosome but not flagellum maturation. Epididymal epithelial cells and luminal fluid from each segment are being examined to better understand the source of cenexin secretion and its incorporation into spermatozoa. Results from these studies will aid in further understanding the physiology of sperm maturation during epididymal transit and increase male fertility preservation options.

Role of Ca2+ in the IVM of spermatozoa from the sterlet Acipenser ruthenus

2017 ◽  
Vol 29 (7) ◽  
pp. 1319 ◽  
Author(s):  
Olga Bondarenko ◽  
Borys Dzyuba ◽  
Marek Rodina ◽  
Jacky Cosson
Keyword(s):  
Sperm Motility ◽  
Seminal Fluid ◽  
Mature Male ◽  
Sperm Maturation ◽  
Wolffian Duct ◽  
Testicular Spermatozoa ◽  
Acipenser Ruthenus ◽  
Sterlet Acipenser Ruthenus ◽  
Motility Parameters

The role of Ca2+ in sturgeon sperm maturation and motility was investigated. Sperm from mature male sterlets (Acipenser ruthenus) were collected from the Wolffian duct and testis 24 h after hormone induction. Testicular spermatozoa (TS) were incubated in Wolffian duct seminal fluid (WDSF) for 5 min at 20°C and were designated ‘TS after IVM’ (TSM). Sperm motility was activated in media with different ion compositions, with motility parameters analysed from standard video microscopy records. To investigate the role of calcium transport in the IVM process, IVM was performed (5 min at 20°C) in the presence of 2 mM EGTA, 100 µM Verapamil or 100 µM Tetracaine. No motility was observed in the case of TS (10 mM Tris, 25 mM NaCl, 50 mM Sucr with or without the addition of 2 mM EGTA). Both incubation of TS in WDSF and supplementation of the activation medium with Ca2+ led to sperm motility. The minimal Ca2+ concentration required for motility activation of Wolffian duct spermatozoa, TS and TSM was determined (1–2 nM for Wolffian duct spermatozoa and TSM; approximately 0.6 mM for TS). Motility was obtained after the addition of verapamil to the incubation medium during IVM, whereas the addition of EGTA completely suppressed motility, implying Ca2+ involvement in sturgeon sperm maturation. Further studies into the roles of Ca2+ transport in sturgeon sperm maturation and motility are required.

METABOLISM OF RAM TESTICULAR SPERMATOZOA IN THE PRESENCE OF TESTOSTERONE AND RELATED STEROIDS

1970 ◽  
Vol 65 (3) ◽  
pp. 565-576 ◽  
Author(s):  
J. K. Voglmayr ◽  
R. N. Murdoch ◽  
I. G. White
Keyword(s):  
Aerobic Glycolysis ◽  
Rete Testis ◽  
Glycolytic Metabolism ◽  
Anaerobic Conditions ◽  
Aerobic Conditions ◽  
Testicular Spermatozoa ◽  
Almost All ◽  
Oxidation Of Glucose

ABSTRACT The effects of testosterone* and related steroids on the oxidative and glycolytic metabolism of freshly collected ram testicular spermatozoa and of spermatozoa stored under air in rete testis fluid for 3 days at 3°C have been studied. When freshly collected testicular spermatozoa were incubated with glucose under aerobic conditions only a small proportion of the utilized glucose could be accounted for as lactate. The addition of a number of steroids, including testosterone, androstanedione, 5β-androstanedione, androsterone, epiandrosterone and 5β-androsterone, greatly increased aerobic glycolysis, the oxidation of the substrate and the proportion of the utilized substrate converted to lactic acid. After 3 days storage at 3°C, testicular spermatozoa respired at a greater rate than spermatozoa freshly collected from the testes. Although the stimulating effect of steroids on aerobic glycolysis increased after storage, they depressed rather than stimulated the oxidation of glucose by stored testicular spermatozoa. With the exception of androstanedione, which slightly stimulated glycolysis, storage of testicular spermatozoa for 3 days in the presence of steroids did not significantly influence their subsequent metabolism when washed free of the steroids. Both freshly collected and stored ram testicular spermatozoa displayed a marked Pasteur effect, and utilized more glucose and produced more lactate under anaerobic than under aerobic conditions. In the absence of oxygen the steroids did not stimulate glycolysis to any extent. However, epiandrosterone depressed the glycolysis of freshly collected spermatozoa under anaerobic conditions and after storage, 5β-androsterone had a similar effect. Androstanedione, 5β-androstanedione, epiandrosterone and 5β-androsterone were the most effective steroids in altering the metabolism of testicular spermatozoa and, under almost all conditions of incubation, depressed the synthesis of amino acids from glucose. The results suggest that the effects of testosterone and related steroids in vitro may depend on the age of the spermatozoa after their release from the Sertoli cells; the steroid effects may have important consequences in vivo in relation to sperm maturation.

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

scholarly journals Grow Smart: An Integrated Nutrition and Early Child Development Intervention Among Infants Improves Expressive Language and Reduces Anemia and Iron Deficiency in Rural India (FS08-03-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Kristen Hurley ◽  
Sylvia Fernandez-Rao ◽  
K Madhavan Nair ◽  
Nangalla Balakrishna ◽  
Kankipati Radhakrishna ◽  
...  
Keyword(s):  
Child Development ◽  
Repeated Measures ◽  
Expressive Language ◽  
Early Learning ◽  
Emotional Behavior ◽  
Rural India ◽  
York Academy ◽  
Developmental Potential ◽  
Visual Reception ◽  
Main Effects

Abstract Objectives Nutritional deficiency and lack of early learning opportunities contribute to the loss of developmental potential. The objective is to evaluate the effects of an integrated nutrition and early learning intervention (ELI) on micronutrient status and the development of infants in rural India. Methods 513 infants (6–12mo) were enrolled from 26 villages in rural India and randomized using a 2 × 2 design to receive multiple micronutrient powders (MNP containing iron, zinc, vitamins A, B2, B12, C and folic acid) vs. placebo (B2) and EL vs. control. The ELI was based on the UNICEF-developed Care for Child Development. Baseline (BL), post-intervention (6 mo) and follow-up (12 mo) evaluations included Mullens Scales of Early Learning, anthropometry, and 2 ml venous blood (BL & 12 mo). Data were analyzed using linear mixed effects regression models with random village/subject intercepts accounting for site of recruitment and repeated measures. For biomarker outcomes, main effects of MNP was assessed. P-values of <0.05 considered statistically significant. For child development outcomes, main effects of MNP and ELI were analyzed separately, and three-way interactions (MNP*ELI*midline or MNP*ELI*end line) were tested. Results At baseline, the mean age of mothers was 22.9y (SD = 2.9) and 99.6% were married. Most mothers (84.2%) had attended some schooling. Mean infant age was 8.6 mo (SD = 2.2); 53.0% were male. Anemia prevalence was 66.4%. No significant baseline differences were found. At end line, infants in the MNP group had significantly higher hemoglobin (11 g/dl vs.10 g/dl) and ferritin (18.2ug/l vs.11.5 ug/l) values compared to infants in the placebo groups, respectively. Infants in the MNP group (mean = 39.5; SE = 0.6) versus placebo group (mean = 37.7; SE = 0.6) also scored significantly higher in expressive language and marginally higher in visual reception (mean = 42.5; SE = 0.5; P = 0.06) and social-emotional behavior (mean = 24.1; SE = 0.2; P = 0.052), compared to the placebo (mean = 41.1; E = 0.6, mean = 23.5; SE = 0.2, respectively). Significant interactions in visual reception and expressive language performance showed that children who received either or both interventions had better scores than children who received neither. Conclusions Home MNP and EL interventions can improve infant MN status and development. Funding Sources Mathile Institute for the Advancement of Human Nutrition, Nutrition International, Sackler Institute for Nutrition Science of the New York Academy of Science.

Sperm maturation in the domestic cat

2006 ◽  
Vol 66 (1) ◽  
pp. 14-24 ◽  
Author(s):  
Eva Axnér
Keyword(s):  
Domestic Cat ◽  
Sperm Maturation
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