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RESEARCH |
Laboratory of Physiology, School of Veterinary Medicine, University of Thessaly, Karditsa, Greece, 1 Laboratory of Physiology, School of Veterinary Medicine, Aristotle University of Thessaloniki, University Campus, 541 24 Thessaloniki, Greece, 2 Laboratory of Physiology, Department of Animal Production, Technological Educational Institute of Epirus, Arta, Greece and 3 Department of Animal Production, Technological Educational Institute, Larissa, Greece
Correspondence should be addressed to I A Taitzoglou; Email: jotai{at}vet.auth.gr
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Introduction |
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ik et al. 1988, De Jonge et al. 1989), the recognition and binding of spermatozoa to the zona pellucida (Saling 1981, Jones 1991), the penetration of the zona pellucida by spermatozoa (Kennedy et al. 1983, Schill 1991), the fusion of the gamete membranes (Tesaik 1995) and development of the block to polyspermy by zona hardening (Smith et al. 2000). There are at least two types of plasminogen activator (PA) in mammals: the tissue-type PA (t-PA) and the urokinase-type PA (u-PA); both convert the abundant extracellular zymogen plasminogen into plasmin, which is the active proteinase that stimulates fibrinolysis and proteolysis. PAs, as well as PA inhibitors (PAIs) and plasmin inhibitors, have been found in ejaculated spermatozoa from various species, sheep included (Smokovitis et al. 1987). The PAs and their inhibitors are localized to the outer acrosomal membrane and plasma membrane of human and boar spermatozoa (Smokovitis et al. 1992). It has been proved that, during the acrosome reaction, PAs are released in human, boar, bull and ram spermatozoa (Taitzoglou et al. 1996), and that plasmin induces the acrosome reaction (Taitzoglou et al. 2003) and influences various motility parameters (Taitzoglou et al. 2004) in bull spermatozoa. A number of studies show a positive correlation between PA activity (PAA) of human spermatozoa and their fertilizing ability (Lison et al. 1993, Liu et al. 1996, Huang et al. 1997).
Vitamin A is required for several critical life processes, including reproduction, metabolism, differentiation, hematopoiesis, bone development, and pattern formation during embryogenesis (Sporn et al. 1994). In the testis, vitamin A is required for the normal production of spermatozoa (Mason 1933). In the seminiferous epithelium of vitamin A-deficient mice, a depletion of germ cells occurs. Only Sertoli cells, type A spermatogonia, and occasionally a few degenerating spermatids are present (van Pelt & de Rooij 1990). Resupplementation of retinol (Morales & Griswold 1987) or retinoic acid (van Pelt & de Rooij 1991) results in the re-initiating of spermatogenesis in a synchronized manner. The mechanism by which retinoids elicit their effects ultimately resides in their ability to regulate gene expression at specific target sites (Mangelsdorf 1994). Several types of retinoid receptor have been identified; among them, the types retinoic acid receptor 
1 (RAR1), RAR
(Kim & Wang 1993) and retinoid X receptor (RXR; Huang et al. 1994) have been identified in the testis. These receptors are active during the whole of spermatogenesis and until the phase of elongating spermatids in spermiogenesis (Dufour & Kim 1999).
It is known that retinoids can affect various parameters of the fibrinolytic system. Indeed, experiments in vitro showed that retinoids increase t-PA synthesis by human endothelial cells (Kooistra et al. 1991), while they enhance t-PA levels in rat tissues in vivo (van Bennecum et al. 1993). Furthermore, retinoids, when supplemented per os, affect various parameters of the human fibrinolytic system (Declerck et al. 1993). On the contrary, to our knowledge the possible effect of vitamin A on acrosin activity has not been investigated to date. Therefore, the present study was undertaken to investigate the effect of excessive vitamin A intake or vitamin A deprivation on the activity of acrosin and PAs of ram spermatozoa, and their possible correlation.
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Blood samples and semen samples were collected weekly. The ejaculated semen was collected using a small ruminant artificial vagina. Immediately after collection, sperm motility was evaluated subjectively by preparing a wet mount of the spermatozoa and estimating the percentage of motile spermatozoa in four fields. Additionally, sperm concentration was determined using a Neubauer hemocytometer. Acrosomal status, viability and percentage of abnormal forms were evaluated using the staining procedure eosin/nigrosin (Thompson & Cummins 1985). Spermatozoa were classified as (1) dead with intact acrosome, (2) dead with detached acrosome, (3) live with intact acrosome and (4) live with detached acrosome. Slides were examined with a bright-field microscope. For classification, microscope fields were selected at random and all spermatozoa in each field were evaluated until 200 spermatozoa per slide were examined.
Acrosin assay
Preparation of solutions was as follows. Ficoll: 11% (w/v) Ficoll was diluted in 0.12 M NaCl/0.025 M Hepes buffer, pH 7.4. Benzamidine solution was made by dissolving a peptidase inhibitor in water to the concentration of 0.5 M. N--benzoyl-DL-arginine paranitroanilide-HCL (BAPNA) extender was made with 0.01% (v/v) Triton X-100 in 0.055 M Hepes/0.055 M NaCl at pH 8.0. BAPNA was diluted in DMSO to a concentration of 23 mM. Immediately before use it was further diluted nine times with BAPNA extender.
Acrosin activity was determined in ram spermatozoa by spectrophotometry (Kennedy et al. 1989). In brief, 5 µl of each ejaculate were placed in 1000 µl NaCl/Hepes, pH 7.4, vortexed, and a volume containing 2 x 106 spermatozoa was washed with Ficoll solution. After rediluting the washed spermatozoa, 0.5 x 106 sperm cells were diluted in NaCl/Hepes to a final volume of 100 µl. 100 µl benzamidine solution were added to control tubes and 1000 µl BAPNA solution were added to all tubes after vortexing. After 90 min incubation at 37 °C, 100 µl benzamidine solution were added to test tubes and all tubes were centrifuged at 1000 g for 15 min. Supernatants were collected and the absorbance was read at 410 nm. The difference between control and test tubes was adopted as net absorbance. Acrosin activity was expressed as µiu 10–6 spermatozoa, as calculated by the formula suggested by Kennedy et al.(1989).
PAA assay
The following solutions were used: 0.73 mM chromogenic substrate solution (S-2251; Chromogenix Milano, Italy); 50% (v/v) acetic acid; and extraction solution prepared by mixing 4 vol 0.1 M Tris/HCl buffer and 1 vol 0.5 M potassium thiocyanate.
Spermatozoa were centrifuged twice with natural saline in order to wash away the seminal plasma. A sample volume containing 1 x 108 spermatozoa was vortexed in 1000 µl extraction solution and centrifuged at 3000 g for 10 min, and supernatants were collected and stored at – 70 °C until assayed.
PAA was determined by spectrophotometry as described in detail by Smokovitis et al.(1987). A total volume of 500 µl mixture consisting of 200 µl substrate solution, 100 µl plasminogen solution (final concentration, 0.5 casein units ml–1), 100 µl Tris/HCl buffer and 100 µl of the acrosomal extract was incubated for 30 min at 37 °C. The reaction was terminated by adding 1000 µl 50% (v/v) acetic acid. The absorbance was read at 405 nm. The difference between control and test tubes was adopted as net absorbance. PAA was expressed as iu t-PA 10–8 spermatozoa, referring to a standard curve designed using 1–50 iu t-PA (Biopool AB, Umea ° Sweden).
Vitamin A determination in blood plasma
Three metabolites of vitamin A (retinoic acid, retinol, and retinyl palmitate) were determined in ram blood plasma using a HPLC method that was specially designed for this project (Moulas et al. 2003). Figure 1
shows typical chromatograms of a standard solution (Fig. 1a) and a ram blood plasma sample (Fig. 1b). In brief, samples of 200 µl were extracted with 2-propanol-dichloromethane, the extracts were centrifuged, and supernatants were collected, evaporated, reconstituted in mobile phase, and analyzed on a C8 column using two consecutive isocratic elutions with methanol tetrahydrofuran-acetate buffer. Detection was performed at 350 and 325 nm using wavelength changes during the run. The method exhibits analytical characteristics well within the acceptable limits. Overall recoveries were 73.7% for retinoic acid, 90.2% for retinol, and 87.7% for retinyl palmitate. Precision values, expressed as the percentage relative standard deviation, were in the range of 1.16–6.18%, while limits of detection were 0.3 ng ml–1 for retinoic acid, 6.0 ng ml–1 for retinol, and 10.0 ng ml–1 for retinyl palmitate.
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). Sperm viability and acrosomal status of control and treated samples showed no statistical differences.
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Due to well-known homeostatic mechanisms, retinol concentration was maintained within the range of 20–50 µg ml–1 during the whole experiment, as suggested by the reviewed literature. The slight increase in retinol concentration during the last few weeks of the experiment should be expected, since rams tend to have such an increase at the onset of the summer (Asadian et al. 1995). On the contrary, retinyl ester and retinoic acid concentrations showed significant changes during the experiment, being non-detectable during vitamin A deprivation, but reaching considerably higher levels after vitamin A resupplementation.
The Sertoli cell is the main site of retinol uptake by the testis. In these cells, vitamin A can be either stored or oxidized to retinoic acid and, after binding to specific nuclear receptors, affect the expression of various genes. Spermatocytes and spermatids also need vitamin A, which is delivered to them by the Sertoli cells essentially as retinol bound to RBP (Davis & Ong 1992) or to cellular RBP (CRBP; Ong 1985), but also as retinyl ester (Kim & Wang 1993) or retinoic acid (Livera et al. 2002). Since spermatocytes and spermatids are protected by the blood–testis barrier, the Sertoli cells are their basic suppliers of vitamin A (Kim & Wang 1993). Nevertheless, during spermatogenesis, from spermatogonia A0 up to the stage of pachytene spermatocytes, germ cells can uptake retinol directly from the blood since they are not protected by the blood–testis barrier. Additionally, retinyl ester, when in high concentrations can diffuse into these germ cells from the blood (Blaner & Olson 1994), and so can retinoic acid (Wolf 2001). It is noteworthy that vitamin A deprivation results in a decrease of RAR in rat testis (Haq et al. 1991), as well as in a transportation of these nuclear receptors to the cytoplasm, where they are inactive (Livera et al. 2002).
In group B, the supplementation of vitamin A at a dose four times higher than the daily needs of the rams, and for 13 weeks, did not affect either enzyme activity. It seems that the supplementation of excessive vitamin A cannot affect the synthesis of these proteolytic enzymes, at least once the needs of the target cells have been covered. On the contrary, acrosin activity and PAA in group C decreased 108 days after vitamin A deprivation, and were kept low for 9 weeks. When retinol concentration in plasma is low, there is also a decrease of CRBP in testis (Rajan et al. 1990). This probably reduces the delivery of retinol to germ cells (Ong 1985). Since retinol is esterified only when bound to CRBP, germ cells receive less retinyl esters as well (Kim & Wang 1993). Finally, CRBP is necessary for the oxidation of retinol to retinoic acid (Blaner & Olson 1994). Since CRBP is not present in spermatocytes or spermatids (Huggenvik & Griswold 1981), it seems that the only source of retinoic acid for these cells are the Sertoli cells (Cavazzini et al. 1996), which produce less retinoic acid in vitamin A deprivation. This results in a decrease of the retinoic acid delivered to the nucleus of the target cell, because delivery of retinoic acid occurs when this is bound to cellular retinoic acid-binding protein (CRABP), and this binding protein acts as a passive vehicle, which binds and releases its ligand according to concentration gradients (Noy 2000).
Retinoic acid, bound to CRABP, modulates nuclear gene expression by binding to specific receptors. These receptors bind as homo- or heterodimers to specific DNA sequences known as retinoic acid-response elements. To date, two such response elements have been identified, direct repeats DR-5 and DR-2, which are found in the promoters of several retinoic acid-responsive genes (Mangelsdorf 1994). At least in humans, DR-5 has been localized on the t-PA gene, and when retinoic acid binds to it the expression of the gene is enhanced (Bulens et al. 1995). On the contrary, no such response element was found on the mouse t-PA gene; this gene and its enhancer activator protein 1 are synergistically activated by cAMP and retinoic acid (Mira-y-Lopez et al. 1998).
During spermatogenesis, the biosynthesis of acrosin takes part in the meiotic phase or immediately after this (Kremling et al. 1991, Raab et al. 1994). A specific time point for the biosynthesis of PAs by the spermatogonia or the spermatids has not been determined. Nevertheless, pachytene spermatocytes and spermatids show an increased t-PA activity during stages IX–XII (Bardin et al. 1994). Therefore, the increased activity of t-PA, the type of PA to which the greatest percentage of PAA of ram spermatozoa is attributed (Smokovitis et al. 1987), coincides with the maximum concentration of CRABP and RAR in germ cells as well.
Apart from direct regulation of gene expression, retinoic acid can also act indirectly, by regulating an intermediary, which in turn regulates the gene of interest, influencing mRNA stability, or activating nuclear receptor dimers other than the RAR–RXR complex (Balmer & Blomhoff 2002). These include other receptors of the steroid receptor superfamily, such as the receptors of vitamin D3 and thyroid hormone (Maden 2000). The two parts of the heterodimer have a synergistic effect on the activation of the target gene (Mangelsdorf 1994). Indeed, a response element on the human t-PA gene has been identified where the heterodimer of the RXR and the vitamin D3 receptor binds and activates its transcription (Merchiers et al. 1999). In fact, it is known that vitamin D3 regulates PA synthesis by normal and malignant osteoblasts (Hamilton et al. 1985), the production of u-PA and PAI-1 by monocytes (Gyetko et al. 1988), and the synthesis of PA by epithelial and fibroblastic cells (Koli & Keski-Oja 1996) or by rat heart microvascular cells (Puri et al. 2000).
Vitamin A deficiency is able to reduce plasma testosterone concentration in rats (Bartlett et al. 1989), and testosterone does affect acrosin activity (Theodosiadou 2000) and PAA (Rekkas et al. 1993). Nevertheless, in the Bartlett et al.(1989) study, the reduction of mean testosterone concentration was observed 112 days after the beginning of a vitamin A-deficient diet, while it is proved that 70–75 days after the supplementation of such a diet to rats spermatogenesis is arrested and the seminiferous epithelium degenerates irreversibly (Ismail et al. 1990). Since sperm counts, motility, and percentage of abnormal forms were not altered during our experiment, while our findings appeared a lot earlier in a species – the sheep – with a definitely greater capacity of storing vitamin A in the liver, we conclude that the possible effect of vitamin A deprivation on any enzyme activity via alterations of testosterone concentration should be excluded.
It is noteworthy that the effect on the activity of either enzyme was shown with a delay of 108 days for vitamin A deprivation and 42 days for vitamin A resupplementation. This is not surprising if we take into consideration that (1) sheep can cover their needs using vitamin A that they have already stored and (2) spermatogenesis in the ram lasts approximately 49 days, followed by the transportation of the spermatozoa in the epididymis for 16 days. This delay possibly supports the suggestion that the effect of vitamin A on acrosin and PAs of spermatozoa is exerted by regulating the biosynthesis of these enzymes during spermatogenesis, rather than the synthesis of PAs, PAIs, or acrosin inhibitors that are taken up by the spermatozoa after they are released into the seminiferous tubule.
In conclusion, excessive vitamin A intake did not affect acrosin or PAA, but vitamin A deprivation reduced the activity of both enzymes in a reverse manner. Recent knowledge about vitamin A metabolism and action in the cell allows us to suggest that it may have influenced acrosin and PA synthesis during spermatogenesis by regulating gene expression, although other, indirect mechanisms of action should not be excluded. To date, the main result of vitamin A deficiency was known to be the arrest of spermatogenesis and testicular degeneration. A new role for vitamin A in the male reproductive system may be suggested, since it can influence factors related to male reproductive ability such as acrosin and PAs, before spermatogenesis is affected.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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indicate statistical differences compared with controls (P < 0.05). Group A (controls), 12 500 iu vitamin A/animal per day; group B, 50 000 iu vitamin A/animal per day; group C, 0 iu vitamin A/animal per day until the 13th week, then 150 000 iu vitamin A/animal per day until the end of the experiment.
, group A (controls); •, group B; 
, 
, group A (controls); •, 
, group B; 