1 Division of Biomedical Sciences, and 2 Academic Department of Obstetrics and Gynaecology and Reproductive Physiology, St Bartholomew's and Royal London School of Medicine and Dentistry, Queen Mary & Westfield College, London E1 4NS, UK
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Abstract |
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Key words: angiotensin II/blood/human/seminal fluid
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Introduction |
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The importance of the RAS in the male is also strongly indicated by the existence of a testis-specific angiotensin converting enzyme (ACE) isozyme that depends on a testis specific promoter in the 12th intron of the ACE gene (Zhou et al., 1995, 1996
). Testis-specific ACE is expressed only in seminiferous tubules and developing spermatozoa (Brentjens et al., 1986
; Mukhopadhyay et al., 1995a
), though earlier reports suggested it is also present in the Leydig cell (Pandey et al., 1984
). In human spermatozoa it has been localized specifically in the plasma membrane of the acrosome, equatorial and post-acrosome regions, and midpiece (Kohn et al., 1998a
). In humans, angiotensin II generation has been demonstrated directly in the testis (Okuyama et al., 1988b
) and both active renin and angiotensin II concentrations in internal spermatic vein plasma were increased in patients receiving HCG (Okuyama et al., 1988a
). Autoradiographic studies, ligand binding assays and immunocytochemistry have all shown that angiotensin II receptors are present in Leydig cells (Millan and Aguilera, 1988
; Vinson et al., 1995a
,b
). In addition, immunocytochemistry reveals that the AT1 receptor is present in cells of the germinal line, including germinal epithelium, and in the tails of developing spermatozoa (Vinson et al., 1995b
).
Further evidence comes from studies on mice with a disrupted gene coding for the somatic and the testis-specific ACE forms. In these, males have reduced fertility, despite having normal testes, spermatozoa and mating behaviour (Krege et al., 1995). Sperm motility was also apparently normal (Esther et al., 1996
), although ACE gene disruption results in poor sperm transport within the oviduct, and poor binding to the zona pellucida (Hagaman et al., 1998b
).
The AT1 receptor identified by immunocytochemistry in human spermatozoa is clearly functional: stimulation of ejaculated spermatozoa by angiotensin II enhances motility and this is inhibited by the specific ATI antagonist losartan (Vinson et al., 1995b, 1996
). Furthermore, the acrosome reaction has been reported to be inhibited by captopril (Foresta et al., 1991
), and there is direct evidence that angiotensin II stimulates acrosomal exocytosis (Muller et al., 1997
; Gur et al., 1998
), though this action has been attributed to the presence of AT1 receptors by some authors (Gur et al., 1998
), but to AT2 receptors by others (Kohn et al., 1998b
). In mouse spermatozoa, local perfusion of motile but non-progressive spermatozoa with angiotensin II evoked a rapid, substantial rise in intracellular [Ca2+], which was blocked by losartan (Wennemuth et al., 1999
). These results are all consistent with the view that spermatozoa possess functional AT(1) receptors that control motility and exocytosis.
If angiotensin II is important to sperm function and fertility, then it is important to evaluate its availability during reproductive events, and during transit through the reproductive tract. Inter alia, it is clearly important to assay angiotensin II concentrations in the ejaculate, not hitherto reported.
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Materials and methods |
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Sperm quality was assessed according to WHO criteria (World Health Organization, 1992). After liquefaction at room temperature for 40 min, 1 ml of seminal plasma was added to centrifuge tubes into the same medium as for blood samples.
Angiotensin II assay
Angiotensin II was assayed in both blood plasma and seminal plasma using a radioimmunoassay kit (Peninsula Laboratories, Merseyside, UK).
Sample extraction
The extraction procedure followed the manufacturers' recommendations. Samples were collected as outlined above and acidified by addition of an equal volume of 1% trifluoracetic acid (TFA) high performance liquid chromatography (HPLC) grade (Merck, Poole Dorset, UK) and centrifuged at 6000 g for 20 min at 4°C. Reverse phase solid phase extraction tubes (Envi-18 containing 500 mg octadecyl, 17% from Supelco, Sigma-Aldrich Company Ltd, Poole, Dorset, UK) were equilibrated with 1 ml acetonitrile and subsequently washed with 3ml of 1% TFA buffer. Samples were loaded onto the pre-treated columns and each column was washed with 6 ml 1% TFA. Angiotensin II peptide fractions were then eluted with 3 ml of 60% acetonitrile in 0.1% TFA and collected in polypropylene tubes. Eluants were evaporated to dryness overnight and stored at 20°C until required.
HPLC
The HPLC system consisted of two pumps (Waters 501; Millipore UK Ltd., Watford, UK), a variable-wavelength ultra violet detector (Waters 486 turntable absorbance detector; Millipore UK Ltd.), an injection system with a 20 µl sample loop, a solvent programmer and a fraction collector.
Separations were carried at room temperature at a flow rate of 1 ml/min on a 300x3.9 mm reversed-phase column (µBondpack C18 Waters 10 µm pore size). An isopropanol gradient (Merck) was used whereby isopropanol buffers contained 10 mmol/l triethylammonium phosphate (TEAP; Merck), pH 3, adjusted with phosphoric acid (Merck), increased from 5 to 40% in a linear fashion over 45 min. Before using the mobile phase buffers, they were degassed under helium gas.
To verify the choice of column and mobile phase, 20 µg of each of the synthetic angiotensin peptides were loaded on the column and peaks detected using UV detection at 220 nm and 0.2 absorbance units full scale. Before each HPLC run, the column was equilibrated with buffer B (40% isopropanol, 10 mmol/l TEAP pH 3) for 10 min followed by buffer A (5% isopropanol, 10 mmol/l TEAP pH 3), also for 10 min.
As the amount of angiotensin II present in plasma was not detectable by UV a mixture of angiotensin peptides was loaded on the HPLC column at a concentration of 100 pg/20 µl, which was within the range of the angiotensin II assay kit, and fractions were collected over an appropriate time scale. Retention times of the synthetic angiotensin peptides were consistent with those published (Hermann et al., 1988), and fraction collections were started when each HPLC run was 30% completed and continued for a period of 15 min. The fraction collector was programmed to collect a sample every 0.5 min. These fractions were subsequently dried overnight and analysed via angiotensin II radioimmunoassay. This provided information on the exact fraction number at which each angiotensin peptide eluted and also indicated the specificity of the commercial angiotensin II radioimmunoassay kit.
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Results |
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Discussion |
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Taken together, there was no correlation between blood and seminal plasma angiotensin II, and for the values shown in Figure 3, the correlation coefficient was 0.11. Inspection of the data suggests there may be subgroups within these samples, however, and notably a small subset has high blood angiotensin II. If the six samples with values >30 pg angiotensin II/ml blood were excluded, there was a significant relationship with seminal plasma angiotensin II (r = 0.72), though the latter values were invariably higher than blood values, by a factor of about two on average. There may also be two groups of high (>50 pg/ml) and low seminal plasma angiotensin II. Although neither of these discriminations was associated with any clinical criterion, the existence of a correlation between the two sets of values, at least in a subgroup of subjects, suggests that there may be some universal systemic regulatory system that affects both, even though seminal plasma values (and thus the seminal plasma responses to such regulation) were higher.
Perhaps remarkably, in view of all the evidence regarding the presence of the testicular and indeed germ cell specific isoform of ACE, and its relationship to fertility (Foresta et al., 1987; Esther et al., 1997
; Hagaman et al., 1998a
), the data presented here do not support the view that spermatozoa themselves contribute greatly to seminal plasma angiotensin II, since oligospermic or aspermic samples nevertheless contained seemingly similar concentrations of angiotensin II as normal samples (Figure 4
). In preliminary results with a small group of 16 patients, no correlation was found between rapid, slow or total forward progressive spermatozoa and seminal angiotensin II. However, bearing in mind that these subjects, in attendance at a fertility clinic, may have a varied aetiology, this is a topic that will need further investigation. In addition, studies on vasectomized patients, or in split ejaculates, would also be valuable to ascertain the possible origins of the hormone.
The most important point, however, is that, taken with evidence of the actions of angiotensin II on sperm motility and the acrosome reaction (Foresta et al., 1991; Vinson et al., 1995b
, 1996
; Muller et al., 1997
; Gur et al., 1998
), it is clear that angiotensin II is not only important for sperm function, it is also available in significant concentrations.
One point this concept raises is that the acrosome reaction, and binding to the ovum, may occur only after the spermatozoa have spent some hours in the uterus (Yanamigachi, 1994). Thus the immediate significance of seminal angiotensin II to these events is less clear. How far seminal plasma angiotensin II can be transported in the vagina and uterus, and for what period it can be maintained at a significant concentration, are questions requiring answers that cannot at present even be guessed at. If it is a reasonable speculation, however, that, like most active hormones in tissue or plasma, the half-life of seminal plasma angiotensin II in the female tract is unlikely to be much more than a few minutes, then the immediate significance of the ejaculated hormone is more likely to be connected with sperm motility, and perhaps capacitation. This does not exclude a critical role for angiotensin II at later stages too, but suggests that other sources of the hormone may then be more important. As in the male, evidence for local RAS has been described for several sites in the female tract (Hsueh, 1988
; Sealey and Rubattu, 1989
; Hagemann et al., 1994
; Vinson et al., 1997
), and, of these, the ovary might perhaps be most pertinent to this discussion. Prorenin production, renin and angiotensin converting enzyme, and the mRNA coding for them, have all been demonstrated in the ovary (Derkx et al., 1987
; Itskovitz and Sealey, 1987
; Bumpus et al., 1988
; Metzger et al., 1988
; Howard and Husain, 1992
). Increased peritoneal angiotensin II concentrations in the peri-ovulatory period suggest that follicular angiotensin II is released at ovulation into the Fallopian tube (Delbaere et al., 1996
). It may be this source of angiotensin that is most relevant to the actions of angiotensin of spermatozoa at the time of ovum fertilization, and also facilitate ovum transport, in view of the presence of angiotensin II AT1 receptors in both Fallopian tube and uterine epithelial cells, as well as in spermatozoa (Vinson et al., 1995a
,b
, 1996
; Delbaere et al., 1996
; Saridogan et al., 1996a
,b
).
The present data also raise the question of the site of synthesis of seminal plasma angiotensin II, given that the contribution of the spermatozoa appears to be slight. While renin and angiotensinogen have both been identified in the testis and epididymis, production rates of angiotensin would seemingly need to be very high to account for the concentrations of the hormone that were detected in seminal plasma, given the great dilution of the testicular product by prostatic secretion. The same would seem to be true for the presence of prorenin in seminal plasma (Mukhopadhyay et al., 1995b). The possibility that the prostate itself might secrete angiotensin would seem to require careful study.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 23, 1999; accepted on March 3, 2000.