Results: 5

1.
Fig. 4.

Fig. 4. From: Delayed testicular aging in pituitary adenylate cyclase-activating peptide (PACAP) null mice.

Comparison of StAR and P450c17 protein levels by Western blots after rescue experiment. A rescue experiment was performed as described in Materials and Methods. StAR and P450c17 protein levels were assessed in two 4-month-old and two 15-month-old PACAP−/− animals that received i.p. injections of 2 nmol of PACAP (+PACAP) and compared with the levels of the same proteins in two 4-month-old and 15-month-old testes from PACAP−/− animals that received i.p. injections of water (+Water) as a control. Shown is the representative intensity of the blots seen for each group of animals.

Arnaud Lacombe, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3793-3798.
2.
Fig. 5.

Fig. 5. From: Delayed testicular aging in pituitary adenylate cyclase-activating peptide (PACAP) null mice.

Schematic representation of putative mechanisms leading to protection against testicular aging in PACAP−/− mice. In wild-type animals (Left), PACAP produced by the hypothalamus stimulates the release of LH at the pituitary level. In testis, PACAP is also synthesized by germ cells. Both PACAP and LH act on Leydig cells, to enhance testosterone biosynthesis. Generation of ROS directly results from steroidogenesis. These free radicals, by inducing apoptosis in testicular cells, alter the architectural integrity of seminiferous tubules and ultimately cause testicular aging. Conversely, in PACAP−/− animals (Right), steroidogenesis is down-regulated in Leydig cells. Consequently, fewer ROS are produced, and germ cell apoptosis is inhibited. Finally, this mechanism leads to a protection against testicular aging in PACAP−/− mice.

Arnaud Lacombe, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3793-3798.
3.
Fig. 3.

Fig. 3. From: Delayed testicular aging in pituitary adenylate cyclase-activating peptide (PACAP) null mice.

Peroxynitrite formation and testicular apoptosis in 15-month-old wild-type and PACAP−/− mice. (A and B) Peroxynitrite formation. Two testis sections of 15-month-old wild-type (n = 3) and 15-month-old PACAP−/− (n = 3) mice were stained with the anti-nitrotyrosine antibody to reveal the degree of peroxynitrite formation. Three different controls were included for each experiment: without the primary antibody, without the secondary antibody, and without primary and secondary antibodies, all showing a complete absence of staining (data not shown). Note the strong staining of peroxynitrites found in the old wild-type testis, in contrast to the moderate staining in the PACAP−/− testis. (Scale bar, 50 μm.) (C and D) Representative examples of apoptotic germ cells detected by the TUNEL method. In situ detection of cells with DNA strand breaks was assessed in three testis sections from 15-month-old wild-type (Left; n = 4) and PACAP−/− (Right; n = 5) mice, as described in Materials and Methods. For negative controls (data not shown), terminal deoxynucleotidyl transferase enzyme was substituted with PBS. Sections were counterstained with methyl green. Black arrows show the localization of apoptotic cells in the seminiferous tubules. (Scale bar, 50 μm.)

Arnaud Lacombe, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3793-3798.
4.
Fig. 1.

Fig. 1. From: Delayed testicular aging in pituitary adenylate cyclase-activating peptide (PACAP) null mice.

Comparison of testosterone and steroidogenesis levels during aging between wild-type and PACAP−/− animals. (A) Serum testosterone levels during aging in wild-type and PACAP−/− males. Between 4 (n = 8) and 15 (n = 8) months of age, serum testosterone decreased dramatically in wild-type animals, whereas levels remain low and constant in PACAP−/− mice (n = 9 for 4-month-old and n = 15 for 15-month-old animals) during the same period. (B and C) Comparison of serum LH (B) and serum FSH (C) levels during aging in wild-type and PACAP−/− males. Levels of LH and FSH hormones were assayed as described in Materials and Methods. For LH, 15 wild-type and 10 PACAP−/− males at 4 months of age and 6 wild-type and 9 PACAP−/− males at 15 months of age were used. For FSH, 16 wild-type and 10 PACAP−/− males at 4 months of age and 7 wild-type and 12 PACAP−/− males at 15 months of age were used. (D and E) Expression profile of StAR and 3β-HSD during aging. Testes of both wild-type (n = 5 for 4- and 8-month-old and n = 3 for 15-month-old) and PACAP−/− (n = 6 for 4-month-old and n = 5 for 8- and 15-month-old) animals were dissected and prepared as described in Materials and Methods. Level of expression of StAR (C) is significantly reduced in wild-type animals when aging but remained low and constant in KO animals. Similar experiments performed with 3β-HSD (D) revealed no significant difference in expression levels in wild-type animals during the aging process. In PACAP−/− mice, similar to that of StAR, the level of expression of 3β-HSD was low and constant over time. Data presented in the graph are the results of two independent real-time quantitative PCRs, performed with individual samples run in triplicate. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.005.

Arnaud Lacombe, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3793-3798.
5.
Fig. 2.

Fig. 2. From: Delayed testicular aging in pituitary adenylate cyclase-activating peptide (PACAP) null mice.

Histopathology of 4- and 15-month-old wild-type and PACAP-deficient mouse testes. (A) Testes of both wild-type mice at 4 (n = 5) and 15 (n = 5) months of age and PACAP-deficient mice at 4 (n = 4) and 15 (n = 8) months of age were fixed in 4% paraformaldehyde and embedded into paraffin. Mounted sections (6 μm) were deparaffinized, rehydrated, and stained with hematoxylin and eosin. (Scale bars, 100 μm.) The black arrow shows the depletion of cells often observed in the seminiferous tubules of old wild-type animals, and the white arrow points to the vacuolizations also found at high frequency around the seminiferous tubules of these animals. Note the good preservation of germ cells in the old PACAP−/− testis compared with the wild type. (Inset) c-kit Staining, confirming the loss of germ cells in the old wild-type testis when compared with the old PACAP−/− testis. (Scale bars, 50 μm.) The associated graph represents the number of seminiferous tubules counted per testis section for the wild-type and PACAP−/− animals. (B) Rate of seminiferous tubule degeneration. On three testis sections stained with hematoxylin and eosin (n between 4 and 8 for each group), two populations of seminiferous tubules were scored as explained in Materials and Methods. Results are presented as the percentage of degenerated seminiferous tubules per testicular section. (C) Testis weight. Testes and epididymis were dissected out and weighed in both wild-type and PACAP-deficient mice at 4 months of age (n = 11 and n = 10, respectively) and also at 15 months of age (n = 9 and n = 7, respectively). ∗∗, P < 0.05; ∗∗∗, P < 0.005.

Arnaud Lacombe, et al. Proc Natl Acad Sci U S A. 2006 March 7;103(10):3793-3798.

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