Tag: Prostaglandin

DISCUSSION(4)

Because enzyme activity of PGDS in the epididymal fluid is unlikely, we investigated its function as a putative androgen and RA transporter. RA was a good candidate, because 1) it is present in the epididymis, 2) it is important for maintenance of the epididymal epithelium, and 3) Tanaka et al. previously demonstrated that recombinant human PGdS is able to bind RA. Our results showed clearly that PGDS binds RA in vitro after purification and in the epididymal fluid. However, saturation of the binding sites was not obtained in the presence of excess ligand, suggesting that the binding is not specific, as it is for several other luminal proteins able to bind RA in vivo.

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Taken together, these results strongly suggest that the enzymatic activity of PGDS is very low (or even absent) in the epididymal lumen, with our experimental conditions being close to in vivo conditions. Previous studies demonstrating the existence of enzymatic activity of epididymal PGDS were performed in drastic conditions (pH 9) that seem incompatible with the epididymal milieu. Moreover, the enzymatic function of PGDS occurs only when the molecule is in its reduced form, but the protein is secreted in vivo with its disulfide bonds.

The secretion of PGD2 in the lumen of the tubule might be related to other luminal pathways, such as the activity of glutathione S-transferase or albumin. Because we observed PGD2 inside and outside the tubule, a more likely hypothesis is that PGD2 might originate from intracellular enzyme activity and then be secreted into the lumen or outside the tubule. The biological effect of PGD2 in the brain is mediated by its specific receptor. This receptor has been identified in the rat epididymis. However, its regionalization is restricted to the caput, whereas PGDS is more abundant in the cauda (unpublished results).

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Several putative sites of glycosylation (N and O) were observed in the protein sequences for both species. We have previously described PGDS isoforms using 2D electrophoresis in the epididymal fluid of rams and stallions. The difference of 8-10 kDa between the theoretical and the apparent molecular mass of PGDS and the presence of at least six spots with a pI range of 4-7 are probably the result of posttransductional modifications at these putative sites. In the cerebrospinal fluid of the human and rat, PGDS is also glycosylated, as is PGDS in the human epididymal fluid.

The enzyme substrate of PGDS is PGH2, which results from the conversion of arachidonic acid by COX activity (COX1 and COX2). COX2 is not expressed in the ram epididymis, although COX2 is present in the rat epididymis, suggesting variation between species. COX1 was detected only in tissue extracts in the ram epididymis and not in the lumen content. COX1 has been shown to be localized on spermatozoa membranes in the bull, but this was not found in the ram. Because COX1 localization is exclusively intracellular in the ram epididymis, the availability of pGh2 (a very labile molecule) for PGDS in the epididymal extracellular fluid is highly unlikely, although PGH2 has been shown to be exchanged between cells.

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DISCUSSION(1)

In this study, we investigated the functional and biochemical characteristics of PGDS, a major protein secreted in the mammal epididymis present in the milieu surrounding the spermatozoa throughout the male genital tract. To evaluate the function of PGDS in male reproduction, we first analyzed the variations in the presence of PGDS in the semen and researched a putative correlation between such variations and male fertility in the ram and bull. Concentrations of PGDS in the semen of both species showed strong variations in immunoblotting between animals. The origin of such variations is unknown. We have previously shown that PGDS in the semen originates from the epididymal fluid (and not from prostatic or seminal secretions). One hypothesis is that these variations might result from variations in epididymal secretion. However, such wide variations in PGDS secretion were not observed in our previous study in the ram and stallion. The variations observed in the semen likely relate to a degradation process of the protein after its secretion in the epididymis.

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To summarize our investigation of this pathway, PGD2 production in the epididymis under our experimental conditions was quantitatively irrespective of the presence of PGDS in the lumen. These results strongly suggest that luminal PGDS is not involved in the enzyme production of PGD2. These results were corroborated by the absence of PGD2 production when PGH2 was directly added to the epididymal fluid and its absence in seminal plasma even in the presence of PGDS cofactors such as DTT, which is reported to be involved in enzyme activity in the brain (data not shown).

Effect of PG pathway inhibition on fertility and spermatozoa mobility using nonsteroidal anti-inflammatory drugs. This study was performed in adult rats. Seven days of daily i.p. treatment with flurbiprofen and indomethacin were sufficient to inhibit synthesis of PGE2 and PGF2 by seminal vesicles (data not shown), suggesting that the doses used were sufficient to inhibit the whole PG pathway in the male genital tract. After 60 days of this treatment, fertility of the males was estimated by breeding with normal females. No differences were observed between treated animals and controls (Table 2). The motility of caudal spermatozoa was unchanged after treatment, except for progressive mobility. These results suggest that inhibition of the PG pathway does not adversely affect male reproduction in the rat.

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Metabolic PG pathway in the epididymis. The presence of COX1 in the ram epididymis indicated that the PG pathway is functional in this organ. To investigate this pathway, we used an in vitro incubation technique with closed-end epididymal tubules as previously described in the presence of [3H]arachidonic acid. The radioactive PGs produced in the luminal fluids, tissues, and supernatants during incubation were detected and quantified as described in Materials and Methods.

In the first experiment (two animals), the PGs synthesized inside and outside the tubules of the caput (E2) and corpus (E6) were analyzed (Fig. 6). The neosynthesized PG profiles were similar for both epididymal regions without any significant qualitative or quantitative difference. For both regions, the same products were detected inside and outside the tubule in relatively similar amounts. The most abundant of the different PGs detected was 6-keto-PGF1a (prostacyclin degradation product). The other major PGs detected were PGE2, PGF2a and its degradation product (13,14-dihydro-15-keto PGF2a), thromboxane B2 (derived from thromboxane A2), and PGD2. In the epididymal caput and corpus (lumen and supernatant), PGD2 represented 710% of the total PGs detected, without any difference outside and inside the tubule.

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RESULTS(3)

Metabolic PG Pathway in the Epididymis

To investigate the putative enzymatic function of PGDS, we performed an overall study of the metabolic PG pathway in the epididymis. The aim of this approach was to determine if this pathway is functional in the epididymis by detecting the cyclooxygenases (COX1 and COX2) in the ram epididymis, analyzing in vitro PG synthesis in isolated epididymal tubules using exogenous radioactive ar-achidonic acid, and evaluating the effect of the pharmacological inhibition of this pathway on fertility in the rat.

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