Identification of diabetes- and obesity-associated proteomic changes in human spermatozoa by difference gel electrophoresis.

Difference gel electrophoresis (DIGE) of fluorescently labelled human sperm proteins was used to identify diabetes- and obesity-associated changes of the sperm proteome. Semen samples from type 1 diabetics, non-diabetic obese individuals and a reference group of clinically healthy fertile donors were evaluated in a comparative study. The adaptation of a general protein extraction procedure to the solubilization of proteins from isolated progressively motile human spermatozoa resulted in the detection of approximately 2700 fluorescent protein spots in the DIGE images. Comparison of the patients' sperm proteomes with those of the reference group allowed the identification of 20 spots containing proteins that were present in the sperm lysates at significantly increased or decreased concentrations. In detail, eight of these spots were apparently related to type 1 diabetes while 12 spots were apparently related to obesity. Tryptic digestion of the spot proteins and mass spectrometric analysis of the corresponding peptides identified seven sperm proteins apparently associated with type 1 diabetes and nine sperm proteins apparently associated with obesity, three of which existing in multiple molecular forms. The established proteomic approach is expected to function as a non-invasive experimental tool in the diagnosis of male infertility and in monitoring any fertility-restoring therapy.

Evaluation of poly(ADP-ribose) polymerase cleavage (cPARP) in ejaculated human sperm fractions after induction of apoptosis.

OBJECTIVE: To examine the presence of cleaved poly(ADP-ribose) polymerase(s) (cPARP) in ejaculated spermatozoa and determine cPARP levels following exposure to chemical or oxidative stress. DESIGN: Prospective pilot study. SETTING: Tertiary care academic hospital. PATIENT(S): Eight healthy men. INTERVENTION(S): Semen specimens were collected, prepared with double-density-gradient centrifugation, and divided into control, hydrogen peroxide (H(2)O(2)), H(2)O(2) + 3-aminobenzamide (3-ABA), staurosporine (STS), and STS + 3-ABA treated groups. MAIN OUTCOME MEASURE(S): Cleaved PARP and apoptosis markers by flow cytometry. RESULT(S): Cleaved PARP was detected in both neat and mature fractions. The cPARP levels were similar in both mature and immature spermatozoa. In combined mature and immature fractions, a higher percentage of late apoptotic sperm was seen in STS + 3-ABA versus STS. Higher levels of late apoptotic spermatozoa were seen in immature versus mature fractions within STS and STS + 3-ABA groups. Lower levels of cPARP were seen in immature versus mature fractions in H(2)O(2) and H(2)O(2) + 3-ABA treated groups. Cleaved PARP was related to activated caspase-3. CONCLUSION(S): Cleaved PARP is present in ejaculated human spermatozoa. Poly(ADP-ribose) polymerase inhibitors may play a different role in chemical versus oxidative stress-induced sperm damage.