Antimicrobial drug ornidazole inhibits hamster sperm capacitation, in vitro

Studying potential dietary exposure to antimicrobial drug residues via meat and dairy products is essential to ensure human health and consumer safety. When studying how antimicrobial residues in food impact the development of antimicrobial drug resistance and disrupt normal bacteria community structure in the intestine, there are diverse methodological challenges to overcome. In this study, traditional cultures and molecular analysis techniques were used to determine the effects of tetracycline at chronic subinhibitory exposure levels on human intestinal microbiota using an in vitro continuous flow bioreactor. Six bioreactor culture vessels containing human fecal suspensions were maintained at 37 °C for 7 days. After a steady state was achieved, the suspensions were dosed with 0, 0.015, 0.15, 1.5, 15, or 150 µg/mL tetracycline, respectively. Exposure to 150 µg/mL tetracycline resulted in a decrease of total anaerobic bacteria from 1.9 × 107 ± 0.3 × 107 down to 2 × 106 ± 0.8 × 106 CFU/mL. Dose-dependent effects of tetracycline were noted for perturbations of tetB and tetD gene expression and changes in acetate and propionate concentrations. Although no-observed-adverse-effect concentrations differed, depending on the traditional cultures and the molecular analysis techniques used, this in vitro continuous flow bioreactor study contributes to the knowledge base regarding the impact of chronic exposure of tetracycline on human intestinal microbiota.

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Minimum and maximum extracellular Ca2+ requirements during mouse sperm capacitation and fertilization in vitro

Reproduction ◽

10.1530/jrf.0.0810077 ◽

1987 ◽

Vol 81 (1) ◽

pp. 77-89 ◽

Cited By ~ 95

Author(s):

L. R. Fraser

Keyword(s):

Fertilization In Vitro ◽

Sperm Capacitation ◽

Mouse Sperm

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Signal transduction mechanisms involved in in vitro ram sperm capacitation

Reproduction ◽

10.1530/rep.1.00770 ◽

2006 ◽

Vol 132 (5) ◽

pp. 721-732 ◽

Cited By ~ 45

Author(s):

Patricia Grasa ◽

José Álvaro Cebrián-Pérez ◽

Teresa Muiño-Blanco

Keyword(s):

Tyrosine Phosphorylation ◽

Calcium Influx ◽

Calcium Entry ◽

Calcium Entry Blocker ◽

Protein Tyrosine Phosphorylation ◽

Sperm Capacitation ◽

Protein Tyrosine ◽

Entry Blocker ◽

Ram Sperm

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca2+, NaHCO3and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca2+. cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.

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Chlortetracycline staining patterns of frozen-thawed bull spermatozoa treated with β-cyclodextrins, dibutyryl cAMP and progesterone

Zygote ◽

10.1017/s0967199400001040 ◽

2000 ◽

Vol 8 (3) ◽

pp. 245-256 ◽

Cited By ~ 16

Author(s):

Michael B. Dinkins ◽

Benjamin G. Brackett

Keyword(s):

Intestinal Mucosa ◽

Sperm Cells ◽

Fluorescent Staining ◽

Sperm Capacitation ◽

Dibutyryl Camp ◽

Bovine Spermatozoa ◽

Differential Responses ◽

Definition Of

Efforts to achieve complete chemical definition of media used for in vitro capacitation of bovine spermatozoa including removal of heparin purified from porcine intestinal mucosa are presented. Fluorescent staining with chlortetracycline (CTC), known to reflect changes coincident with sperm capacitation in certain species, was studied following treatments of frozen-thawed bull spermatozoa with β-cyclodextrins, dibutyryl cAMP (dbcAMP) and progesterone in comparison with heparin. The CTC staining patterns (F, B and AR) were confirmed to correlate with known conditions that effectively prepare cryopreserved bull spermatozoa for fertilisation in vitro. In the absence of glucose, the routinely employed heparin-containing capacitating medium caused an increase in spermatozoa displaying the AR pattern. Both progesterone (100 μM) and dbcAMP (0.01–0.1 mM) were able to increase the proportion of B pattern stained sperm cells more than after exposure to control (mDM) conditions without a significant reduction in motility. Exposure to either dbcAMP or β-cyclodextrins was accompanied by an increase in proportions of spermatozoa displaying the AR pattern over those seen in controls. Exposure to β-cyclodextrins did not increase the proportion of B pattern stained spermatozoa. Comparison of spermatozoa from two bulls revealed differential responses of spermatozoa from different males to treatments with heparin and progesterone. In vitro fertilisation results demonstrated that previously cryopreserved bull spermatozoa could be capacitated in chemically defined conditions devoid of heparin or other biological components.

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SPERM CAPACITATION BY UTERINE FLUID OR BETA-AMYLASE IN VITRO

Obstetrical & Gynecological Survey ◽

10.1097/00006254-196604000-00039 ◽

1966 ◽

Vol 21 (2) ◽

pp. 322-325

Author(s):

KENNETH T. KIRTON ◽

HAROLD D. HAFS

Keyword(s):

Sperm Capacitation ◽

Uterine Fluid

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Biological effects of polyphenol-rich extract and fractions from an oenological oak-derived tannin on in vitro swine sperm capacitation and fertilizing ability

Theriogenology ◽

10.1016/j.theriogenology.2017.12.015 ◽

2018 ◽

Vol 108 ◽

pp. 284-290 ◽

Cited By ~ 9

Author(s):

Marcella Spinaci ◽

Vera Muccilli ◽

Diego Bucci ◽

Nunzio Cardullo ◽

Beatrice Gadani ◽

...

Keyword(s):

Biological Effects ◽

Sperm Capacitation ◽

Fertilizing Ability

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Penicillamine prevents ram sperm agglutination in media that support capacitation

Reproduction ◽

10.1530/rep-15-0413 ◽

2016 ◽

Vol 151 (2) ◽

pp. 167-177 ◽

Cited By ~ 8

Author(s):

T Leahy ◽

J P Rickard ◽

R J Aitken ◽

S P de Graaf

Keyword(s):

Sperm Capacitation ◽

Dibutyryl Camp ◽

Phosphorylated Proteins ◽

Bicarbonate Ions ◽

Merocyanine 540 ◽

Highly Effective ◽

Lactate And Pyruvate ◽

Ram Sperm ◽

Sperm Population

Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7±2.7% to 2.8±1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.

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Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation

Development ◽

10.1242/dev.121.4.1129 ◽

1995 ◽

Vol 121 (4) ◽

pp. 1129-1137 ◽

Cited By ~ 6

Author(s):

P.E. Visconti ◽

J.L. Bailey ◽

G.D. Moore ◽

D. Pan ◽

P. Olds-Clarke ◽

...

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Tyrosine Phosphorylation ◽

Bovine Serum ◽

Female Reproductive Tract ◽

Dependent Manner ◽

Protein Tyrosine Phosphorylation ◽

Sperm Capacitation ◽

Protein Tyrosine

The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

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The Ubiquitin-Proteasome System Does Not Regulate the Degradation of Porcine β-Microseminoprotein during Sperm Capacitation

International Journal of Molecular Sciences ◽

10.3390/ijms21114151 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4151

Author(s):

Lucie Tumova ◽

Michal Zigo ◽

Peter Sutovsky ◽

Marketa Sedmikova ◽

Pavla Postlerova

Keyword(s):

Seminal Plasma ◽

Plasma Proteins ◽

Protein Composition ◽

Surface Protein ◽

Ubiquitin Proteasome System ◽

Sperm Capacitation ◽

Sperm Surface ◽

Ubiquitin Proteasome ◽

Seminal Plasma Proteins

Sperm capacitation, one of the key events during successful fertilization, is associated with extensive structural and functional sperm remodeling, beginning with the modification of protein composition within the sperm plasma membrane. The ubiquitin-proteasome system (UPS), a multiprotein complex responsible for protein degradation and turnover, participates in capacitation events. Previous studies showed that capacitation-induced shedding of the seminal plasma proteins such as SPINK2, AQN1, and DQH from the sperm surface is regulated by UPS. Alterations in the sperm surface protein composition also relate to the porcine β-microseminoprotein (MSMB/PSP94), seminal plasma protein known as immunoglobulin-binding factor, and motility inhibitor. MSMB was detected in the acrosomal region as well as the flagellum of ejaculated boar spermatozoa, while the signal disappeared from the acrosomal region after in vitro capacitation (IVC). The involvement of UPS in the MSMB degradation during sperm IVC was studied using proteasomal interference and ubiquitin-activating enzyme (E1) inhibiting conditions by image-based flow cytometry and Western blot detection. Our results showed no accumulation of porcine MSMB either under proteasomal inhibition or under E1 inhibiting conditions. In addition, the immunoprecipitation study did not detect any ubiquitination of sperm MSMB nor was MSMB detected in the affinity-purified fraction containing ubiquitinated sperm proteins. Based on our results, we conclude that UPS does not appear to be the regulatory mechanism in the case of MSMB and opening new questions for further studies. Thus, the capacitation-induced processing of seminal plasma proteins on the sperm surface may be more complex than previously thought, employing multiple proteolytic systems in a non-redundant manner.

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