3 METODOLOGÍA
3.2.3 INGENIERÍA DEL PROYECTO
3.2.3.2 Capacidad de producción de la planta
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Fig. 3.1. W estern blotting o f crude extracts from adult rat testis. The western blot shows results from different gels, run under reducing conditions. Molecular weights of marker proteins run with the lysates are given in kDa, migration o f the marker proteins is indicated by arrowheads. After incubation with P2X, receptor (lane 1), P2X^ (lane 3), P2X5 (lane 5) and PZX^ (lane 7) 2 bands o f approximately 70 kDa and 140 kDa were
detected; immunoreactivity for P2Xo receptors (lane 2) was seen at four different bands
of approximately 30 kDa, 42 kDa, 70 kDa and 140 kDa; no immunopositivity for P2X4
receptors (lane 4) and P2X,, (lane 6) was detected.Omission of the primary antibody (lane 0) led to absence o f immunoreactivity.
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Fig. 3.2. Im m u n oreactivity fo r P 2X receptors on b lood vessels o f ad u lt rat testes. Fresh frozen testes o f adult rats were processed for the immunodetection o f P2X receptors and staining for P2X receptors was developed with the D A B/nickel method. (A) blood vessel immunoreactive for P2X, receptors. (B) a magnified part o f the blood vessel stained for P2X, in (A). (C) an area corresponding to the vessel in (B) on an adjacent section was preabsorbed for P2X, antibody. (D) immunostaining for P2Xo receptors in a blood vessel (arrowheads), adjacent to two sem iniferous tubules (arrows), which also show P2X? receptor immunostaining.
P2X Receptors in Seminiferous Tubules.
Fig. 3.3 and 3.4 give an overview on the distribution o f P2X-immunoreactive cells in the adult rat testis.
No staining for P2Xi receptors was observed in the seminiferous tubules. Immunostaining for P2%^ receptors was detected in cells close to the basement membrane o f the seminiferous tubules and in cells o f the adluminal region o f the seminiferous epithelium during stages I to VIII o f the cycle o f the seminiferous epithelium (Fig. 3.3.A, 3.3.B, 3.3.C). In a section o f a seminiferous tubule at stage VII incubation with preabsorbed P2X2 antibody resulted in complete absence o f staining
(Fig. 3.3.D). Infrequently myoid cells surrounding the seminiferous tubules were immunostained for P2X2.
Staining for P2Xg receptors was also detected in areas close to the basement membrane, and in adluminal parts o f the seminiferous tubules during stages I to VIII. Spermatids at stages VI, VII and VIII could clearly be seen at lower magnification (compare Fig. 3.3.E). Preabsorption o f the P2X3 antibody with its cognate peptide
resulted in complete loss o f staining, as shown in Fig. 3.3G on the adluminal part o f a tubule in stage VII o f the cycle o f the seminiferous epithelium.
Immunoreactivity for P2X4 receptors was not seen on any tissue sample.
Incubation with antibody to P2X5 receptors showed staining o f germ cells in stages
IX to XIII (Fig. 3.4.A ), being most intense at stage XII and also gave staining in some myoid cells surrounding the tubules. Staining for P2X5 could be completely preabsorbed
(Fig. 3.4.B).
for P2Xé receptors. At lower magnification staining for P2X7 receptors was only detected in the basal compartment o f the seminiferous tubules and was present throughout all stages o f the cycle o f the seminiferous epithelium (Fig. 3.4.C). Higher magnification o f P2X7-immunopositive cells revealed their location close to the basement
membrane o f the tubules in normal light microscopy (Fig. 3.4.D) and in phase contrast microscopy (Fig. 3.4.E). Preabsorption o f the P2X7 antibody resulted in complete absence o f staining (Fig. 3.4.F).
Fig. 3.3. Overview of P2X receptors in seminiferous tubules of adult rat testes. The distribution o f immunoreactivity for P2X receptors using the DAB/nickel technique is shown. (A) Immunoreactivity for P2X2 receptors in a seminiferous tubule at stage III o f the cycle. Cells in the peripheral area o f the tubule and round and elongated structures in the adluminal parts o f the seminiferous epithelium are stained. (B) Immunoreactivity for P2X2 receptors at stage VII, showing immunostaining o f cells in the basal compartment and in the adluminal region. (C) The sickle shaped structures in the luminal part o f the tubule in (B) at higher magnification. (D) Staining is absent after a preabsorption experiment for P2X2 receptors in a seminiferous tubule at stage VII. (E) Immunostaining for P2X] receptors in sickle shaped luminal structures, and in elongated structures located in the adluminal compartment. Staining for P2X] receptors is shown in a tubule at stage VI (double arrows), and in a tubule at stage VII (arrow). (F) A magnified area o f the adluminal part o f the tubule at stage VI in (E). (G) Staining for P2Xg receptors was completely abolished after preabsorption o f the antibody (shown in, adluminal area o f a tubule at stage VI). Scale bars: A, B and E 80 pm; C, D, F and G 30 pm.
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- V ir , t t ' X. uFig. 3.4. Overview of P2X receptors in seminiferous tubules of adult rat testes. (A) Immunoreactivity for P2X$ receptors in cells located adluminally within the seminiferous epithelium in a tubule at stage X. (B) Immunostaining for P2X$ receptors was completely abolished after preabsorption in a seminiferous tubule at stage X. (C) The distribution o f immunoreactivity for P2Xv receptors in various adjacent tubules is restricted to the outer border o f the tubules. (D) normal light microscopy, and (E) phase contrast microscopy, o f immunostaining for P2Xy receptors close to the walls o f tw o adjacent seminiferous tubules. (F) Absence o f immunostaining for P2X7 receptors after preabsorption on a consecutive slide in an area corresponding to D. Scale bars: A, B and C 80 pm; D, E and F 30 pm.
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V. . '*• 4 # % . \ • * 'Identification o f P2X-immunopositive cells and stages within the cycle o f the seminiferous epithelium.
At higher magnification cells showing immunostaining for P2X receptors (with the plain DAB method) could be identified by counterstaining with haematoxylin (Fig. 3.5 and Fig. 3.6). The results o f the identification o f P2X-immunopositive cells in the seminiferous tubules are summarised in Tab. 3.1.
Immunoreactivity for P2X2 receptors was found in the Sertoli cell perinuclear region throughout stage I to stage VIII o f the cycle, being weaker at stages I, II, VI, VII and VIII, and being more intense at stages III, IV and V. Staining for P2X2 receptors in
developing gametes could be seen at various stages. Staining for P2X2 receptors appeared
on the cell membranes o f type A spermatogonia and pachytene sperm atocytes throughout stages I to VIII. Type B spermatogonia were labeled through stages IV to VI and preleptotene spermatocytes were stained for P2X2 receptors through stages VI to
VIII. In step 2 through step 8, spermatids staining for P2X2 receptors appeared to be in
the area o f the developing acrosome, seeming most intense at step 3. Late step 16 and early and late step 17 spermatids (stages III to V o f the cycle o f the seminiferous epithelium) were intensely labeled for P2X2 receptors. The immunolabeling for P2X2
was most abundant when the spermatids moved close to Sertoli cell nuclei and sometimes continuous labeling was detected between the Sertoli cell perinuclear region and groups o f late step 16 and step 17 spermatids. Intense staining for P2X2 was also
seen on the convex site o f step 18 to late step 19 spermatids (Fig. 3.5.A).
Staining for P2X] receptors closely resembled the findings described above for P2X2
stages I to VIII o f the cycle o f the seminiferous epithelium, the labeling being most abundant at stages III, IV and V. Type A spermatogonia and pachytene spermatocytes were immunoreactive for P2X3 receptors through stage I to VIII. Type B spermatogonia
were labeled for P2X3 receptors from stage IV to stage VI and preleptotene
spermatocytes were stained for P2X3 from stage VI through stage VIII. The developing acrosoma o f step
2
to step 8 spermatids labeled forP2X3
receptors, the staining being most abundant at step 3. Throughout stages III to V o f the cycle o f the seminiferous epithelium late step 16, early step 17 and late step 17 spermatids were intenselyimmunopositive for P2X3 (Fig. 3.5.B). The most intense staining for P2X3 receptors was
found in spermatids o f step 18, and early and late step 19 at the convex site o f the spermatids (Fig. 3.5.C).
Fig. 3.5. Identification o f P2X receptor imm unopositive cells in testes o f adult rats. The identity o f P2X receptor immunoreactive cells could be revealed using higher magnification and the plain DAB method for colour precipitation and light counterstaining with haematoxylin. (A) Immunostaining for P2X2 receptors in a section o f
a seminiferous tubule at stage VII. The preleptotene spermatocytes (PI) in the peripheral area o f the tubules are strongly immunopositive for P2X2 receptors; Pachytene
spermatocytes (P) are more weakly labeled (double arrows); round shaped step 7 spermatids (7) in the adluminal part o f the seminiferous epithelium label for P2X2
receptors in the area o f their developing acrosome (arrows); the elongated step 19 spermatids (19) in the luminal part o f the tubule are stained at their convex side
(arrowheads), (B) Immunostaining for P2X] receptors in a section at stage III. The
approximate location o f the basement membrane is indicated by a dotted line; the perinuclear region o f a Sertoli cell (S) is labeled for P2Xg receptors (arrowheads); type A spermatogonium (A) and some pachytene spermatocytes (P) are labeled on their cytoplasmic membranes; the area o f the developing acrosome in (round shaped) step 3 spermatids (3) is stained for P2X] receptors (arrows); elongated step 16 spermatids (16,
double arrows) are labeling for P2X3 receptors as they move closer to the perinuclear
region o f a Sertoli cell (arrowheads). (C) Immunolabeling for P2X3 receptors at the convex
side o f elongated, luminal step 19 spermatids (19) . Scale bars: 20 pm.
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Staining for P2X$ receptors was seen in pachytene spermatocytes through stage X to stage XII o f the cycle o f the seminiferous epithelium, and in the diplotene spermatocytes o f stage XIII. Immunoreactivity for P2X$ receptors on spermatids was detected from stage X to stage XIII, being most intense at stage XII. A t stage X, spermatids (step 1 0) o f the more adluminal parts o f the seminiferous tubule were immunonegative for P2X$, but became progressively more stained for P2X5 as they moved towards the perinuclear region o f the Sertoli cells (Fig. 3.6.A). Labeling for P2X$
was seen in pachytene spermatocytes, and in step 1 0 spermatids close to the periphery
o f the seminiferous tubule, but the adluminal step 10 spermatids were unstained (Fig. 3.6.A).
Immunoreactivity for P2Xy receptors was seen in Sertoli cells throughout all stages o f the cycle o f the seminiferous epithelium. Fig. 3.6.B shows two Sertoli cells
immunolabeled for P2X? receptors at stage V. Other cell types like type A
spermatogonia, step 5 spermatids and step 17 spermatids were not immunostained. The only other structure displaying staining for P2Xy is the concave site o f late step 19 spermatids (Fig. 3.6.C).
Fig. 3.6. Identification o f P2X receptor imm unopositive cells in testes o f adult rats. (A) Incubation with antibody for P2X$ receptors in a section at stage X; the dotted line indicates approximate location o f the basement membrane; Sertoli cells (S) and leptotene spermatocytes (L) are unlabeled for P2X$ receptors, whereas pachytene spermatocytes (P) are strongly stained; elongating step 10 spermatids (10) in the luminal part o f the tubule are unstained, but step 1 0 spermatids that already moved closer to the perinuclear region o f Sertoli cells become immunolabeled (arrows). (B) Immunostaining for P2X? receptors in a section at stage V; the dotted line outlines the approximate location o f the basement membrane; several cell types unstained for P2Xy receptors are visible: ty p e A spermatogonia (A), pachytene spermatocytes (P), round step 5 spermatids (5) and elongated step 17 spermatids (17) are all unlabeled. Sertoli cells (S) show staining for P2X7 receptors in their perinuclear region. (C) Immunostaining for P2X? receptors at the concave side o f step 19 spermatids (19). Scale bars: 20 yim.