Vitamin A deficiency leads to an arrest of spermatogenesis and a loss of advanced germ cells in male mice. In the present study, the effects of several retinoids and carotenoids on these mouse testis ...were investigated. First, the proliferative activity of the growth-arrested A spermatogonia in vitamin A-deficient (VAD) mice testis was determined, 20, 24, or 28 h after administration of 0.5 mg all-trans-retinoic acid (RA). The bromodeoxy-uridine (BrdU) labeling index of A spermatogonia in control VAD testis was 5 +/- 1% (n = 4, mean +/- SD). When RA was injected (ip), the highest labeling index was found 24 h after RA administration; 49 +/- 5%. When various concentrations of RA, all-trans-4-oxo-retinoic acid (4-oxo-RA) or all-trans-retinol acetate (ROAc), ranging from 0.13-1 mg, were injected, the labeling index of A spermatogonia always increased in comparison with the VAD situation. A maximum index at 24 h was found when 0.5 mg 4-oxo-RA was injected: 56 +/- 3%. This labeling index was even higher than those after injection of RA or ROAc, 49 +/- 5% and 34 +/- 6% respectively. The increase of the BrdU labeling index was dose dependent. After an initial increase of the labeling indices with increasing retinoid doses, the labeling indices decreased at a higher concentration. This decrease is likely due to a concentration dependent timeshift of the optimum of BrdU labeling to shorter time intervals after retinoid administration because a labeling index of 66 +/- 1% was found 20 h after injection of 1 mg RA. At 24 h, this labeling index was halved: 33 +/- 2%. These indices show that the degree of synchronization of spermatogenesis is also dependent on the retinoid dose. When the dimers of RA and 4-oxo-RA, respectively beta-carotene (beta C) and canthaxanthin, were given, 24 h after administration BrdU-labeling indices comparable with the VAD value were found. Repeated injection of beta C twice a week did induce a reinitiation of spermatogenesis, but compared with RA, the activity of beta C was lower and delayed. It is concluded that 4-oxo-RA is active in adult mammals in vivo. It is at least as potent as RA in the induction of the differentiation and subsequent proliferation of growth-arrested A spermatogonia in VAD mice testis. Furthermore, the degree of synchronization of spermatogenesis is influenced by the retinoid dose. Finally, carotenoids were shown to act in the induction of spermatogonial cell proliferation too but with a lower and delayed activity.
Retinoid X receptors (RXRs) are key regulators in retinoid signaling. Knowledge about the effects of 9-cis-retinoic acid (9-cis-RA), the natural ligand for the RXRs, may also provide insight in the ...functions of RXRs. In this study, the effect of 9-cis-RA on spermatogenesis in vitamin A-deficient (VAD) mice was examined. Administration of 9-cis-RA stimulated the differentiation and subsequent proliferation of the growth-arrested A spermatogonia in the testis of VAD mice. However, compared with all-trans-retinoic acid (ATRA), relatively higher doses of 9-cis-RA were necessary. This could not simply be due to a lower or delayed activity of 9-cis-RA, as simultaneous administration of ATRA and 9-cis-RA did not cause a synergistic effect. Instead, the presence of 9-cis-RA diminished the effect of ATRA by approximately one third. Studies of in vivo transport and metabolism showed that ATRA and 9-cis-RA, after administration to VAD mice, penetrated the testis equally well. However, 9-cis-RA was metabolized much faster than ATRA, and other metabolites were formed. This may account for the above-described differential effects of ATRA and 9-cis-RA on spermatogenesis. Similar to ATRA, 9-cis-RA transiently induced the messenger RNA expression of the nuclear RA receptor RAR beta, suggesting a role for this receptor in the effects of retinoids on the differentiation and proliferation of A spermatogonia. In contrast, the messenger RNA expression of the nuclear retinoid receptors RXR alpha, -beta, and -gamma was not changed significantly by administration of their ligand, 9-cis-RA. Hence, 9-cis-RA does not seem to exert its effect on spermatogenesis through altered expression of the RXRs.
The testicular gene expression of the retinoic acid receptors, RAR alpha, -beta, and -gamma, was studied in normal mice and in vitamin A-deficient mice after the administration of all-trans-retinoic ...acid (ATRA). All three types of RARs were expressed in normal and/or vitamin A-deficient testes. Only the expression of RAR beta messenger RNA was transiently induced within 24 h after ATRA injection. ATRA-induced RAR beta expression was also found in purified Sertoli cells, suggesting that these cells mediate at least part of the effect of retinoids on germ cells. When an equimolar amount of retinol was administered instead of ATRA, no induction of RAR beta was seen at the point of maximal induction by ATRA, suggesting that the effect of retinol was delayed and probably less. The related nuclear receptors, RXR alpha, -beta, and, for the first time, gamma, were also shown to be present in the mouse testis. Upon administration of ATRA, messenger RNA expression of RXR alpha and -beta did not change significantly. The expression of RXR gamma was too low to allow quantification. Finally, the effect of the retinoid metabolism inhibitor liarozole on ATRA-induced proliferation of A spermatogonia was examined. The labeling index of A spermatogonia, 24 h after the administration of 0.25 mg ATRA, was significantly lowered by liarozole due to a shift of the maximal 5-bromo-deoxyuridine incorporation to an earlier point (20 h). This indicates that liarozole delays retinoid metabolism, thereby increasing the actual ATRA concentration, and more importantly, that ATRA by itself is an active retinoid in spermatogenesis. Apparently, ATRA does not need to be metabolized to 4-oxo-RA, which was previously shown to be a more potent inducer of spermatogonial proliferation than ATRA, to be effective.
Retinoid X receptors (RXRs) are key regulators in retinoid signaling.
Knowledge about the effects of 9-cis-retinoic acid
(9-cis-RA), the natural ligand for the RXRs, may also
provide insight in the ...functions of RXRs. In this study, the effect of
9-cis-RA on spermatogenesis in vitamin A-deficient (VAD)
mice was examined. Administration of 9-cis-RA stimulated
the differentiation and subsequent proliferation of the growth-arrested
A spermatogonia in the testis of VAD mice. However, compared with
all-trans-retinoic acid (ATRA), relatively higher doses
of 9-cis-RA were necessary. This could not simply be due
to a lower or delayed activity of 9-cis-RA, as
simultaneous administration of ATRA and 9-cis-RA did not
cause a synergistic effect. Instead, the presence of
9-cis-RA diminished the effect of ATRA by approximately
one third.
Studies of in vivo transport and metabolism showed that
ATRA and 9-cis-RA, after administration to VAD mice,
penetrated the testis equally well. However, 9-cis-RA
was metabolized much faster than ATRA, and other metabolites were
formed. This may account for the above-described differential effects
of ATRA and 9-cis-RA on spermatogenesis.
Similar to ATRA, 9-cis-RA transiently induced the
messenger RNA expression of the nuclear RA receptor RARβ, suggesting
a role for this receptor in the effects of retinoids on the
differentiation and proliferation of A spermatogonia. In contrast, the
messenger RNA expression of the nuclear retinoid receptors RXRα,
-β, and -γ was not changed significantly by administration of their
ligand, 9-cis-RA. Hence, 9-cis-RA does
not seem to exert its effect on spermatogenesis through altered
expression of the RXRs.
The lateral elements (LEs) of synaptonemal complexes (SCs) of the rat contain major components with relative electrophoretic mobilities (Mr s) of 30000-33000, which are the products of a single gene. ...After one-dimensional separation of SC proteins on polyacrylamide-SDS gels, these components show up as two major bands, whereas upon two-dimensional electrophoresis they are resolved in at least 24 spots, which focus at pH 6.5 to 9.5. In this paper we show that these spots represent phosphorylation variants. For the analysis of the phosphorylation of the 30000- to 33000-Mr SC components during progression through meiotic prophase, we developed a procedure for isolation of fractions of testicular cells of the rat that are enriched in separate stages of meiotic prophase. Analysis of the 30000- to 33000-Mr SC components in these fractions by two-dimensional electrophoresis and immunoblotting showed that phosphorylated variants of the 30000- to 33000-Mr SC proteins occur throughout meiotic prophase. However, the extent of phosphorylation changes between early and mid-pachytene, when one phosphate group is probably added to each of the variants.
The testicular gene expression of the retinoic acid receptors, RARα,
-β, and -γ, was studied in normal mice and in vitamin A-deficient
mice after the administration of all-trans-retinoic acid
(ATRA). ...All three types of RARs were expressed in normal and/or vitamin
A-deficient testes. Only the expression of RARβ messenger RNA was
transiently induced within 24 h after ATRA injection. ATRA-induced
RARβ expression was also found in purified Sertoli cells, suggesting
that these cells mediate at least part of the effect of retinoids on
germ cells. When an equimolar amount of retinol was administered
instead of ATRA, no induction of RARβ was seen at the point of
maximal induction by ATRA, suggesting that the effect of retinol was
delayed and probably less.
The related nuclear receptors, RXRα, -β, and, for the first time,γ
, were also shown to be present in the mouse testis. Upon
administration of ATRA, messenger RNA expression of RXRα and -β did
not change significantly. The expression of RXRγ was too low to allow
quantification.
Finally, the effect of the retinoid metabolism inhibitor liarozole on
ATRA-induced proliferation of A spermatogonia was examined. The
labeling index of A spermatogonia, 24 h after the administration
of 0.25 mg ATRA, was significantly lowered by liarozole due to a shift
of the maximal 5-bromo-deoxyuridine incorporation to an earlier point
(20 h). This indicates that liarozole delays retinoid metabolism,
thereby increasing the actual ATRA concentration, and more importantly,
that ATRA by itself is an active retinoid in spermatogenesis.
Apparently, ATRA does not need to be metabolized to 4-oxo-RA, which was
previously shown to be a more potent inducer of spermatogonial
proliferation than ATRA, to be effective.
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