INVESTIGATION OF PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE IN HUMAN ANDROLOGICAL FLUID AND ITS EFFECT ON SPERM MOTILITY
Brubel R.
a, Kiss P.
a, Vincze A.
a, Reglodi D.
a, Tamás A.
a, Varga A.
a, Varnagy A.
b, Bodis J.
b, Mark L.
c, Hashimoto H
d, Jambor E.
c, Koppan M.
bDepartments of
aAnatomy,
bObstetrics and Gynaecology,
cBiochemistry and Medical Chemistry, University of Pecs, Hungary, Department of Pharmacology, Osaka University, Japan
INTRODUCTION
Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional and pleiotropic neuropeptide. Although PACAP was first described as a hypothalamic peptide influencing the functions of the pituitary gland, the peptide has numerous other biological effects.
Among others, it influences several reproductive functions, from germ cell development, reproductive hormone production, implantation (Isaac and Sherwood, 2008) and fertilization (Tanii et al. 2011). PACAP is known to influence spermatogenesis and to promote fertilization, possibly due to increased motility (Vaudry et al. 2009). It has been shown that PACAP6-38 decreases sperm motility in rodents (Gozes et al. 1998). However, there is relatively limited information available about its actions and occurrence in human semen and its effect on sperm motility.
Mass spectrometry is a high throughput technique to analyze peptide/protein composition of biological fluids. The aim of the present study was (i) to investigate the occurrence of PACAP in human seminal fluid. We also investigated the effects (ii) on human sperm motility following PACAP treatment using computer assisted semen analysis (CASA), and (iii) the sperm morphology of PACAP deficient mice.
MATERIALS AND METHODS
Samples were collected according to approved institutional protocols (3117/2008, 3610/2009). Seminal fluid samples were collected from healthy male volunteers after at least three days of abstinence (age between 25-35, n=10).
Mass spectrometry: Peptidase inhibitor aprotinin (Trasylol) was added to all of the samples prior to mass spectrometry analysis. Matrix assisted laser desorption/ionisation spectrometry was performed to detect the characteristic peak of PACAP38 in the samples.
Motility analysis: 8 µl semen was dropped to the plate and computerized microscopic analysis was performed. One measurement was done without PACAP, while PACAP (100 nM) was also administered to investigate its effects on motility in the second measurement of the same sample.
Sperm Cell morphology: Epididymis was cut out from each PACAP-deficient and wild type animal. Sperms were dissolved in IVF solution, and dropped to a gelatin coated slide. After drying in a 40 oC plate haematoxilin-eosin staining was performed.
Sperms were fotographed, size of sperm head diameter was measured and statistical analysis (non-parametric, Mann-Whitney U-test) was carried out with GraphPad Prism 5.0.
RESULTS
4534.6
0 1000 2000 3000 4000
2500 3000 3500 4000 4500 5000
m/z
4467.9
4421.7
4534.6
50
100
150
200
250
300
Int. [a.u.]
4400 4500 4600 4700 4800 4900
m/z
4473.2 4421.8
4618.8
200 400 600 800
Int. [a.u.]
4400 4500 4600 4700 4800 4900
m/z
MALDI TOF spectrum of PACAP38 standard indicating the molecular weight at 4534.6 Da.
MALDI TOF spectrum of seminal fluid samples. The peptide peak characteristic for PACAP38 (4534.6 Da) could be identified in 50% of the samples (A), while it was missing from the other half of the samples (B).
A B
SEMINAL FLUID PACAP38 STANDARD
Representative pictures of two different semen samples. (A) represents very good results with exceptional motility and concentration, (B) represents average result of fertile semen.
A
B
PACAP38 administration increased the ratio of rapid progressivesperm cells (type A) and decreased the ratio of the other groups (type B, C).
The present results show that PACAP increases the ratio of rapid progressive sperm cells, indicating that the peptide influences the distribution of sperm cells in favor of more motile type.
PACAP could be shown in half of the seminal fluid samples, and the altered sperm morphology of PACAP deficient mice also indicates an important endogenous function. However, further investigation is needed to clarify the exact physiological role and the correlation between the presence of PACAP and possible pathological conditions.
This work was supported by Hungarian National Scientific Grants OTKA T061766, K72592, F67830, CNK 78480 and PTE-MTA „Lendület” research Grant, SROP-4.2.2/B-10/1-2010-0029
CONCLUSIONS
Haematoxilin-eosin stained wild-type (WT, A) and PACAP-deficient (PACAP-/-, B and C) sperms. A: normal sperm from WT mouse. B: Sperm from PACAP-/- mouse, which has smaller head in diameter than the WT. C: Sperm with abnormal head morphology in PACAP-/-. There is a significant difference (P<0,0001) between the size of sprem head diameter. Mean diameter values: 4,14 in PACAP-/- and 5,66 in wild type (WT).
A B
Illustration: Sperms
