Descripción de funciones y actividades por puesto de trabajo

Specificity o f the antibodies.

The specificity o f our antibodies against P2X receptors was previously proven on western blots using lysates from cells transfected with P2X receptors (Oglesby et al.

1999). In the current study the specificity o f our antibodies for P2X receptors was tested by western blotting o f crude extracts from testes o f adult rats. The molecular weights for P2Xi, P2X3, P2X$ and P2Xy receptors were estimated as approximately 70

kDa and 140 kDa. These findings resemble the results obtained by immunoblotting for P2X receptors o f crude extracts o f adult rat thymus (Glass et al. 2000). Immunoblotting for P2X2 receptors gave a different result, displaying two additional bands o f

approximately 30 kDa and 42 kDa. A molecular weight band for P2X% receptors in the range o f 40 kDa was reported from immunoblots o f crude extracts from rat cardiac and smooth muscle (Worthington et al. 1999). In the current study immunoreactivity for P2X4 receptors was absent from the adult rat testis in both histochemical and

immunoblotting experiments. However, in a previous study from our group it was shown by northern blotting that mRNA for P2X^ receptors is expressed in rat testis (Bo et al. 1995), w ith the abundance o f a second, smaller band next to the 1.9 kB band obtained for full-size P2X4 mRNA,. A smaller-sized P2X4 mRNA may point to the

occurrence o f a splice variant o f the P2X4 receptor in the rat testis. Splice variants for P2X4 receptors were repeatedly found (Le et al. 1997;Townsend-Nicholson et al. 1999).

A splice variant lacking the 15 amino acids o f the carboxy terminus o f the receptor (the antigenic sequence o f our antibodies) would not be detected by the antibodies used

in this study. Tanaka et al. (1996) reported the expression o f P2X4 mRNA by in situ

hybridisation. This group used a mixture o f probes against three different epitopes o f P2X4 mRNA, leaving it open which specific epitope o f the receptor they detected.

P2X receptors on blood vessels.

Immunoreactivity for both P2X% and P2X2 receptors was present in vascular

smooth muscle o f the adult rat testis. This is consistent with previous studies, showing the expression o f P2X2 as well as P2Xi receptors in vascular smooth muscle o f several vessels (Nori et al. 1998). The autonomic innervation in the testes o f most mammals was shown to be composed mainly o f excitatory noradrenergic fibers, supplying predominantly the blood vessels (Bell 1972). ATP was shown to be a cotransmitter in noradrenergic nerves and to participate in the control o f blood flow (Bumstock 1990b).

P2X receptors on developing gametes and Sertoli cells.

P2X2 and P2Xg receptors were both expressed in the same cell types and at the

same stages o f the cycle o f the seminiferous epithelium and they were both present in the same steps o f spermatid development. It is well established that P2X2 and P2Xg receptors are coexpressed in sensory neurones (Cook et al. 1997;Vulchanova et al. 1997) and can form heteromultimeric ion channels (Lewis et al. 1995). Our immunohistochemical data suggest that heteromultimeric P2X2/3 receptors may also be

present in testicular cells.

In the present study differential staining for P2X receptor subtypes during the testicular maturation o f germ cells was shown. The changes in receptor expression occurred homogeneously within a group o f germ cells o f the same developmental stage. The abundance o f certain P2X receptors on one homogeneous group o f cells, and the

absence o f P2X receptors on a different group o f cells would separate these two groups with respect to their purinergic signaling. Since several developmental stages o f germ cells are known to occur parallel to each other in the seminiferous epithelium (Leblond and Clermont 1952), a specific mechanism to control and co-ordinate different groups o f germ cells in their development may be indicated. A cell type and maturation stage specific receptor expression, as shown for P2X receptors in this study, could account for co-ordinated purinergic signaling within one homogeneous group, separating it (in respect to purinergic signaling) from a different group not expressing P2X receptors.

Lalevée et al. (1999) speculated upon a pathway for ATP release, experimenting with immature rat Sertoli cell cultures. These authors describe a possible ATP release from Sertoli cells after stimulation with follicle stimulating hormone (FSH) and o f ATP release by germ cells. Filippini et al. (1994) and Meroni et al. (1998a) showed in cultured rat Sertoli cells, that extracellular ATP has profound effects on FSH responsiveness o f Sertoli cells. Through the experiments o f Filippini et al. (1994) and o f Rudge et al. (1995) using the murine Sertoli cell-line TM4 it was shown that purinergic signalling can be mediated by the P2Y2 receptor (P2U receptor in these articles). Foresta et al. (1995)

found that P2X receptors may also participate in the purinergic signaling o f rat Sertoli cells. Taking together the evidence from these previous articles with the findings o f this report, it is speculated that purinergic signaling in the testis can be mediated through P2X and P2Y receptors. Purinergic signaling may act in concert with hormones (like FSH) that are well known to be o f importance in spermatogenesis. It seems unlikely that neuronal release o f ATP is involved in control o f sperm development since the testicular sympathetic innervation in m ost mammals is restricted to blood vessels and

the capsule (Bell 1972). Carvalho et al. (1993) could not find a role for sympathetic innervation in the control o f sperm development since there are no changes after chemical sympathectomy o f adult rats.

P2X2 and P2X3 receptors were strongly expressed in developing germ cells until

stage VIII o f the cycle. Profound changes take place at stage VIII, as the mature generation o f sperm is released (Leblond and Clermont 1952) and Sertoli cells change their hormone responsiveness at this stage, especially their responsiveness to FSH (Parvinen et al. 1980). It is speculated that this change in FSH-responsiveness o f Sertoli cells could account for changes in the magnitude o f ATP secretion firom Sertoli cells, according to the mechanism suggested by (Lalevée et al. 1999). In the subsequent stages (IX-XIV) P2X2 and P2Xg receptors were absent from the tubules and P2X$ receptors

were detected through stages X-XIII. At stage X o f the cycle o f the seminiferous epithelium step 1 0 spermatids start to elongate and move from the adluminal

compartment o f the epithelium to the basal compartment (Leblond and Clermont 1952;Hess 1990b;Hess 1990b). At this stage immunoreactivity for P2Xg receptors was shown in basal, but not adluminar spermatids (Fig. 3.6.A).

Staining for P2X receptors was not detected on the interstitial Leydig cells. This is in contrast to the findings o f Foresta et al. (1996a), who reported P2X7 receptors (P2Z receptors in their article) on cultured rat Leydig cells after pharmacological measurement. However, when cultured rat Leydig cells were immunostained for P2X receptors, a broad expression o f P2X receptor subtypes was detected (Glass R, unpublished observations). Thus, there seem to be some differences in P2X receptor expression in vivo and in vitro.

acrosome o f spermatids. It was shown that during acrosome formation the majority o f proteins synthesised in the endoplasmic reticulum (ER) are transported via the Golgi network to the acrosome located in the perinuclear region (reviewed by Toshimori, 1998). The immunopositve, perinuclear area probably consists o f the ER, the Golgi network and the acrosome, since the stained structure remains lobular in shape, whereas the acrosome itself flattens during development (Toshimori 1998). For human spermatozoa, ATP-gated ion channels can trigger the acrosome reaction (Foresta et al. 1996b) and incubation o f human sperm with ATP increases in vitro fertilization rates (Rossato et al. 1999).

Staining for P2X2 and P2Xg receptors was also found on the convex site o f

spermatids that are released at step 19. This staining occurs in a region where ‘tubulobulbar complexes’ have their origin. These tubulobulbar complexes are thought to be important to sperm-anchoring before release (Russell and Clermont 1976) and in cell volume reduction o f spermatids (Russell 1979).

Staining for P2X7 receptors was observed on the concave site o f step 19 spermatids.

The greatest decrease in spermatid cell volume (about 70%) occurs between step 18 and step 19 spermatids (Russell 1979). This is thought to be achieved mainly through the movement o f spermatid cytoplasm through tubulobulbar complexes into the Sertoli cell cytoplasm. At stage V ll and V lll o f the cycle the apical parts o f Sertoli cells surround the step 19 spermatids and retain them in the adluminal region o f the tubule. The transport o f cytoplasm from mature spermatids into the Sertoli cells takes place at the concave site o f the step 19 (Russell 1979). P2X7 receptors can form membrane pores allowing the passage o f relatively large molecules - up to 900 Da (Steinberg 1987;

Steinberg 1987;Surprenant et al. 1996). Thus, it could be speculated that P2Xy receptors form pores between step 19 spermatids and Sertoli cells through which the aqueous component o f the spermatid cytoplasm could be removed.

In conclusion it was shown that differential, stage-dependent immunostaining for P2X receptors is present in testes o f mature rats. These results suggest that purinergic signaling via P2X receptors may take part in the control o f coordinated germ cell maturation. P2X receptors could also participate in the mediation o f cell volume reduction and spermatid release. However, functional studies will be needed to specifically address the role o f P2X receptors in the testis.

CHAPTER IV.

Immunohistochemical identification of cells expressing ATP-