OBJECTIVETo study the effect of the split of sperm nuclei on the yield of RNA from human sperm.

METHODSHuman sperm were purified by two sequential centrifugations through 40:80 discontinuous gradients of Percoll. Human leukocytes separated from peripheral blood were used as the control. Total RNAs from purified sperm and leukocytes were extracted with both TRIzol and RNeasy Kit. The RNAs from equal number of cells were reverse-transcribed, and quantified by the levels of beta-ACTIN mRNA, which were evaluated by real time polymerase chain reaction.

RESULTSTRIzol failed to digest majority of sperm nuclei even the incubation time was prolonged to 1 h, while no sperm nucleus was found under the light microscope after 1 min digestion with RLT buffer of the RNeasy Kit. Both reagents can digest the nuclei of human leukocytes well. The amount of RNA per 10(6) sperms isolated with RNeasy Kit (149.8+/-24.5 ng) was 4-fold higher (P=0.01) than that extracted with TRIzol (35.5+/-4.0 ng per 10(6) spermatozoa; n=3). The similar yields of the leukocyte RNAs extracted with RNeasy Kit and TRIzol [(765.5+/-229.8) and (958.8+/-201.0) ng per 10(6) cells respectively; n=3, P=0.168] excluded the possibility of different efficacy of these two reagents in RNA isolation.

CONCLUSIONThe split of sperm nuclei is important to the yield of RNA in the human sperm RNA extraction. The nucleus may be the major area for human sperm RNA repositories.

Cell Nucleus ; chemistry ; Humans ; Male ; Polymerase Chain Reaction ; RNA ; analysis ; RNA, Messenger ; analysis ; Spermatozoa ; chemistry

OBJECTIVETo investigate the progesterone-binding site on the normal fertile human sperm membrane after 2 hours of in vitro capacitation.

METHODSViable spermatozoa were selected by a swim-up method. After 2 hours of in vitro capacitation, multipoint saturation binding experiments were performed. Sperm suspension and increasing concentrations of progesterone-11alpha-glucuronide-[125I] iodotyramine (125I-P) were added to 7 total binding tubes respectively, and equal amounts of sperm suspension and 125I-P were added to another 7 corresponding non-specific binding tubes in the presence of 10 micromol/L progesterone. After incubation for 1 hour at 4 degrees C, the radioactivity of both the tubes and the pellets after centrifugation was measured respectively. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were calculated using the mathematical model of single site multi-point saturation method of Scatchard function and least-squares regression.

RESULTSKd was (0.61 +/- 0.04) nmol/L and Bmax was (830 +/- 344) sites/cell. The significance test of the regression equation indicated that r = -0.980, P < 0.01.

CONCLUSIONThere is a high affinity and low capacity binding site for the progesterone (progesterone receptor) on the normal fertile human sperm membrane.

Adult ; Cell Membrane ; chemistry ; Humans ; Male ; Progesterone ; Radioligand Assay ; Receptors, Progesterone ; analysis ; Sperm Capacitation ; Spermatozoa ; chemistry

OBJECTIVESTo investigate the localization and positive percentage of progesterone receptor (PR) on the human sperm surface.

METHODSAfter in vitro capacitation, progesterone binding sites on the sperm were quantitatively analyzed by fluorescence microscopy and flow cytometry using fluorescein isothiocyanate-labeled bovine serum albumin-progesterone complex (P-BSA-FITC).

RESULTSThe spermatozoa stained by P-BSA-FITC mainly showed two labeling patterns, with the green fluorescence on the whole acrosomal region or the equatorial acrosomal region only and the stainless postacrosomal and tail regions. The percentage of progesterone-binding sperm was (30.2 +/- 2.4)%.

CONCLUSIONSThere is selective expression of PR on the human sperm acrosome surface.

Adult ; Cell Membrane ; chemistry ; Flow Cytometry ; Humans ; Male ; Microscopy, Fluorescence ; Receptors, Progesterone ; analysis ; Spermatozoa ; chemistry

Sexual reproduction is marked by the fusion of the sperm cell with the oocyte during fertilization to produce the diploid zygote, in which the lipids in the sperm plasma membrane play an important role. Due to the loss of most cell organelles and DNA transcription, spermatozoa lack protein expression and vesicular transport. However, the lipids of the sperm plasma membrane undergo complicated dynamic changes, which may facilitate the capacitation, binding with zona pellucida, acrosome reaction and fusion of the sperm cell with the oocyte. This paper summarizes the progress in the studies of the lipids in the sperm plasma membrane, their composition, structure, peroxidation, metabolism and role in fertilization.
Acrosome Reaction ; Animals ; Cell Membrane ; chemistry ; Fertilization ; Humans ; Male ; Membrane Lipids ; metabolism ; Sperm Capacitation ; Spermatozoa ; chemistry

Spermatogenesis is a complex developmental process in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. During spermatogenesis, membrane remodeling takes place, and cell membrane permeability and liquidity undergo phase-specific changes, which are all associated with the alteration of membrane lipids. Lipids are important components of the germ cell membrane, whose volume and ratio fluctuate in different phases of spermatogenesis. Abnormal lipid metabolism can cause spermatogenic dysfunction and consequently male infertility. Germ cell membrane lipids are mainly composed of cholesterol, phospholipids and glycolipids, which play critical roles in cell adhesion and signal transduction during spermatogenesis. An insight into the correlation of membrane lipids with spermatogenesis helps us to better understand the mechanisms of spermatogenesis and provide new approaches to the diagnosis and treatment of male infertility.
Cell Adhesion ; Cell Membrane ; chemistry ; Cholesterol ; chemistry ; Glycolipids ; chemistry ; Humans ; Infertility, Male ; Male ; Membrane Lipids ; chemistry ; Phospholipids ; chemistry ; Signal Transduction ; Spermatogenesis ; Spermatozoa ; cytology

The damage to sperm DNA is one of the most important causes of male infertility. Some sperm with damaged DNA may escape from the sperm surveillance mechanism and transmit the damage to the offspring. So research on the damage to sperm DNA has become one of the hot spots in reproductive medicine. The factors that would damage sperm DNA include oxidative stress, microelements, reproductive toxic substances, radioactive rays, and so on, while the body depends on the compressed sperm DNA and anti-oxidation system for the protection of the integrity of sperm DNA. Some drugs such as anti-oxidant, black tea extract, etc, may help to improve and rebuild these protective mechanisms.
Antioxidants ; pharmacology ; DNA Damage ; DNA Repair ; physiology ; Humans ; Male ; Oxidative Stress ; Spermatozoa ; chemistry ; ultrastructure

The sperm DNA integrity is important for the success of natural or assisted fertilization, as well as normal development of the embryo, fetus and child. Some investigations have demonstrated that when the damaged spermatozoa DNA of the husband exceeds than 30%, the couple has very low potential of natural fertility. Several methods are available for the assessment of sperm DNA integrity, which is considered to be a better marker of male fertility potential than conventional semen parameters. Furthermore, monitering sperm DNA integrity may become an important part of the evaluation for couples seeking assisted reproductive techniques such as intracytoplasmic sperm injection (ICSI). Finally, the effects of cryopreservation and preparation techniques on sperm DNA integrity are summarized.
Chromatin ; chemistry ; Cryopreservation ; DNA Damage ; Humans ; Infertility, Male ; etiology ; genetics ; Male ; Reproductive Techniques ; Spermatozoa ; ultrastructure

It is well known that mycoplasma can cause infection in the male reproductive tract. Some studies indicate that Ureaplasma urealyticum (Uu), a species of mycoplasma, is associated with male infertility. Sulfogalactosylglycerolipid(SGG) is the major mammalian male germ cell glycolipid, synthesized via sulfation of galactosylglycerolipid in early primary spermatocytes. Some experiments have proved that SGG is implicated in sperm-egg binding by linking arylsulfatase A (AS-A), SGG's ligand on the egg. SGG can be desulfated by binding mycoplasmas and transformed galactosylglycerolipid, which doesn't bind AS-A. So the binding and degradation of the sperm SGG by mycoplasmas may play a role in the induction of male infertility. As a kind of mycoplasma, Uu can also bind SGG, which offers another explantion for the association of Uu infection with male infertility caused by Uu infection.
Female ; Galactolipids ; physiology ; Humans ; Infertility, Male ; etiology ; Male ; Mycoplasma Infections ; complications ; Sperm-Ovum Interactions ; Spermatozoa ; chemistry

AIMTo establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender.

METHODSSemen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris mTTE synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1 %, 3 %, 5 %, 10 % and 15 % [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity.

RESULTSThe post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P0.05) for 5 % glycerol (42.95 +/- 2.55 and 50.39+/- 2.42, respectively) than those of the other groups (1%: 19.19 +/- 3.22 and 24.84 +/- 3.64; 3%: 34.23 +/- 3.43 and 41.37 +/- 3.42; 10%:15.68 +/- 2.36 and 21.39 +/- 3.14; 15%: 7.47 +/- 1.44 and 12.90 +/- 2.18). The parameters for 30 min equilibration(42.95 +/- 2.55 and 50.39 +/- 2.42) were better (P0.05) than those of the other groups (10 min: 31.33 +/- 3.06 and 38.98 +/- 3.31; 60 min: 32.49 +/- 3.86 and 40.01 +/- 4.18; 90 min: 31.16 +/- 3.66 and 38.30 +/- 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity.

CONCLUSIONCynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.

Animals ; Cryopreservation ; Cryoprotective Agents ; Glycerol ; chemistry ; Macaca fascicularis ; Male ; Semen Preservation ; Spermatozoa ; cytology

Both the primed in situ (PRINS) and the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) techniques constitute alternatives to the conventional (fluorescence in situ hybridization, FISH) procedure for chromosomal investigations. The PRINS reaction is based on the use of a DNA polymerase and labeled nucleotide in an in situ primer extension reaction. Peptide nucleic acid probes are synthetic DNA analogs with uncharged polyamide backbones. The two procedures present several advantages (specificity, rapidity and discriminating ability) that make them very attractive for cytogenetic purposes. Their adaptation to human spermatozoa has allowed the development of new and fast procedures for the chromosomal screening of male gametes and has provided efficient complements to FISH for in situ assessment of aneuploidy in male gametes.
Aneuploidy ; Humans ; Male ; Peptide Nucleic Acids ; chemistry ; Primed In Situ Labeling ; methods ; Spermatozoa ; metabolism