| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Laboratory of Nutritional Biochemistry, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan
b Faculty of Agriculture, Utsunomiya University, Mine-machi, Utsunomiya-shi, 321-8505, Japan
ABSTRACT
The effects of retinoic acid on the development of reproductive organs and egg production in female Japanese quail (Coturnix coturnix japonica) were investigated. Female quail were fed a diet containing retinoic acid at 4 mg/kg (RA) or two diets containing retinyl acetate at 5000 IU/kg (VA1) or 14 000 IU/kg (VA2) after being fed a vitamin A-free diet for 2 wk (experiment 1). The oviduct and ovary grew more rapidly (P < 0.05) in RA-treated quail than in VA-treated quail at 5 wk of age. In addition, the body weight of RA-fed quail was also greater (P < 0.05) than that of VA-fed quail at 5 wk. The RA-treated quail laid their first eggs approximately 5 days earlier (P < 0.05) than the VA-treated quail. Furthermore, these RA-fed quail laid more eggs (P < 0.05) than those VA-fed quail during the experimental period. To confirm the results of experiment 1, a similar experiment was conducted to record the first egg and total eggs laid by quail fed VA2 or RA (experiment 2). The early onset of oviposition was again observed in the RA-treated group (P < 0.01). These results suggest that retinoic acid has a stimulating effect on the reproductive system of female Japanese quail, as has been previously shown in the reproductive system of male Japanese quail.
female reproductive tract, ovary, oviduct
INTRODUCTION
Vitamin A (retinol) is crucial for normal growth, vision, reproduction, maintenance of numerous tissues, and overall survival in many species. In the reproduction system, morphological changes and functional defects occur in animals receiving a vitamin A-deficient diet. In rats, these alterations consist of testicular atrophy, reduction of germinal epithelium [1], and fetal death or abnormalities [2]. Likewise, in chickens, ingestion of a vitamin A-deficient diet by mature cocks resulted in reduced testicular size, loss of spermatids, and degeneration of seminiferous epithelium [3]; in mature hens, egg production was decreased [4]. These lesions can be reversed by administration of vitamin A, demonstrating the importance of this vitamin in the reproductive system.
In vertebrates, most symptoms of vitamin A deficiency can be reversed by administration of retinoic acid, a vitamin A metabolite converted from retinol. However, early studies revealed that retinol-deficient, retinoic acid-fed rats failed to reproduce [5]. Histological examination of the testes in these animals revealed lesions similar to those observed in retinol-deficient rat. Therefore, it has been claimed that retinoic acid cannot replace retinol, both for vision [6] and in reproduction.
The existence of two distinct classes of nuclear receptors for retinoic acid (retinoic acid receptors, RAR
, RARß, and RAR
; and retinoid X receptors, RXR, RXRß, and RXR), which act as ligand-inducible transcriptional regulators, has been described recently [7–9]. Expression of these receptor genes has been observed in the reproductive system [10]. Furthermore, RAR [11] and RXRß [12] null mutant mice are sterile because of severe degeneration of the germinal epithelium or abnormal spermatogenesis, and RAR [13] null mutant mice are also sterile because of squamous metaplasia of the seminal vesicles and prostate. Interestingly, the morphology of the testes in these mice was similar to that observed in the male mice fed the vitamin A-deficient diet. Furthermore, compound null mutations of RAR genes led to embryonic lethality and numerous developmental abnormalities [14, 15] that resembled those observed in the fetal vitamin A-deficient syndrome [16]. Thus, these results strongly suggest that retinoic acid, serving as the active form of vitamin A, is an important signaling molecule that, acting via its nuclear receptors, alters gene expression at the level of transcription and plays a critical role in vertebrate development and the reproductive system.
Recently, we were first to report, to our knowledge, that Japanese male quail fed diets depleted of vitamin A but supplemented with retinoic acid exhibited faster growth of their testes than controls fed vitamin A [17]. This suggests that retinoic acid has an accelerative effect on testicular maturation in quail. To determine whether retinoic acid exerts the same effect on maturation of the female reproductive system as it does on the male, we examined the effect of retinoic acid on development of the oviduct and ovary and on egg production in Japanese quail (Coturnix coturnix japonica).
MATERIALS AND METHODS
Animals and Experimental Treatments
Diets were prepared as described elsewhere [17]. Briefly, a basal diet consisted of isolated soybean protein, glucose monohydrate, rapeseed oil, and the other nutrient requirements, except for vitamin A (Table 1). The three experimental diets were prepared by taking 1 kg of this basal diet and then adding either 5000 IU (VA1) or 14 000 IU (VA2) of all-trans-retinyl acetate (vitamin A; Wako Pure Chemicals, Osaka, Japan) or 4 mg (RA) of all-trans-retinoic acid (retinoic acid; Sigma, St. Louis, MO). Diets were prepared every week and stored at -20°C until use. One-day-old female Japanese quail were housed in a brooder, maintained on continuous illumination for the first 3 days of age, and then subjected to a 14L:10D photoperiod with lights-on at 0500. Diets and water were available at all times throughout the experiment. Fresh diets were provided three times every day to avoid oxidation and esterification of vitamin A. The experimental designs and animal management were reviewed and approved by the Committee of Utsunomiya University for Experimental Animals.
|
Experiment 1 Four-hundred 1-day-old female Japanese quail were fed the basal diet for 2 wk, and then 156 were selected for uniform body weight and allotted to 12 cages of 13 birds each, with four replicated cages, assigned to each VA1, VA2, or RA treatment. Two birds were randomly selected from each cage at 3, 4, and 5 wk of age, weighed individually, and then killed by decapitation. The oviduct and ovary were removed, and their weights were recorded. Plasma and livers were also collected for determining vitamin A levels. Plasma and livers were also collected from newly hatched quails and from 1- and 2-wk-old quail fed the basal diet to assess the degree of vitamin A depletion.
At 5 wk of age, all remaining birds were transferred to individual cages in a temperature-controlled room (25 ± 2°C) and maintained on their respective diets for the duration of the experiment. The experiment continued until birds were 62 days of age. The age and body weight at first egg were recorded for each bird. The first egg and the eggs laid during the entire experimental period were also weighed.
Experiment 2 A similar experiment was performed to confirm the results of experiment 1. Forty-four female quail fed the basal diet for 2 wk from hatching were selected, divided into two groups of 22 birds each, and maintained in individual cages from 5 wk of age as described for experiment 1. One group of birds was fed VA2; the other one was given RA. Age and body weight at first egg were recorded for individual birds. The first egg and the eggs laid during the entire experimental period were also weighed. The experiment was continued until birds were 57 days of age.
Determination of Vitamin A Levels
Retinol levels in the plasma and retinyl palmitate levels in the livers from all collected samples in experiment 1 were determined by HPLC as described elsewhere [18, 19], because the main retinoid in plasma is retinol and that in the liver is retinyl palmitate.
Statistical Analysis
Mean weights for the whole body, oviduct and ovary were analyzed by one-way ANOVA. Total egg weights and total number of eggs per bird during the entire experimental period for experiments 1 and 2 were also analyzed by one-way ANOVA. Post hoc analysis was performed with the Tukey multiple comparison test. The accumulated numbers of quail that had laid their first eggs by each day were analyzed using the chi-square test.
RESULTS
Experiment 1
Changes of vitamin A levels in the plasma and livers of the RA group and the two VA groups are presented in Figure 1. The retinol level in plasma and the retinyl palmitate level in the livers declined rapidly when quail were fed the basal diet for 2 wk, and they continued to decline when quail were fed RA thereafter. In contrast, plasma retinol levels were quickly restored to the initial level within 1 wk and then remained stable when vitamin A-containing diets were given from 2 wk of age. Different vitamin A levels of the diets used in this experiment did not influence plasma retinol levels. Hepatic retinyl palmitate levels also recovered gradually by feeding vitamin A and reflected the dietary vitamin A levels.
|
Body, oviduct, and ovarian weights during development are given in Figure 2. The body weights (Fig. 2A) were not different among the dietary treatments at 3 and 4 wk of age. At 5 wk of age, quail receiving RA had significantly heavier body weights than those receiving VA1 or VA2 (P < 0.05). No significant difference in body weights was found between VA1- and VA2-fed birds.
|
The oviducts of RA-fed quail were heavier at 5 wk of age (P < 0.01) than those of VA1- and VA2-fed quail, but no significant difference was observed at 3 and 4 wk of age (Fig. 2B). The ovaries of RA-fed quail were heavier than those of VA1- and VA2-fed birds at 4 wk (P < 0.05) and at 5 wk (P < 0.01) of age (Fig. 2C). Differences in the oviduct and ovarian weights when corrected for body weight between RA and VA1 treatments or between RA and VA2 treatments were also significant at 5 wk of age (P < 0.01; data not shown). No significant differences were found in the oviduct and ovarian weights between the VA1 and VA2 treatment groups at the same age (Fig. 2, B and C).
The oviduct and ovarian weights at 5 wk of age suggested that the gonad growth of RA-fed quail was advanced, and this idea was confirmed by observations on oviposition. Figure 3 clearly shows the differences in the onset of oviposition between the RA-treated group and the two VA-treated groups. The RA-fed quail laid eggs earlier than those of both VA-treated groups. For example, RA-treated quails began to lay eggs on Day 38, and half of those birds laid their first eggs by 43 days of age. However, half the birds fed VA1 and VA2 laid their first eggs by 48 and 49 days of age, respectively. When the number of birds that had laid their first eggs were compared, significant differences between the RA and VA2 treatment groups were seen on Day 43 (P < 0.01) and Day 49 (P < 0.05) and between the RA and VA1 treatment groups on Day 43 (P < 0.05) and Day 49 (P < 0.05).
|
The age and mean body weights at first egg laying and the egg yield during the entire experimental period are summarized in Table 2. The ages when 50% of the birds were laying eggs were 43 days in the RA-treated group versus 48 and 49 days in the VA1- and VA2-treated groups, respectively. Birds commenced egg laying when their body weights reached approximately 120 g in all treatment groups. The mean weight of the first eggs did not differ among the treatment groups. However, RA-treated quails (63.9 g/bird) laid more eggs (P < 0.05) than VA1-treated (45.5 g/bird) and VA2-treated birds (46.0 g/bird) during the whole experimental period.
|
Experiment 2
To confirm the results observed in experiment 1, we conducted a similar experiment to record the first egg and the total eggs laid by quail fed VA2 or RA. The onset and frequency of oviposition are presented in Figure 4. The early onset of oviposition was again observed in the RA-treated group. By 43 days of age, 11 of 22 RA-fed birds and 2 of 21 VA2-fed birds laid their first eggs, with the difference being significant (P < 0.01). Moreover, by 50 days of age, the number of birds that had laid the first eggs in the RA-fed group (19 of 22) was significantly greater (P < 0.01) than that in the VA2-fed group (11 of 21). The RA-treated quail (58.5 g/bird) also laid more eggs (P < 0.05) than the VA2-treated birds (38.0 g/bird) during the entire experimental period. Therefore, the stimulating effect of retinoic acid on the onset of oviposition has been confirmed.
|
DISCUSSION
In the present report, the effects of retinoic acid on the development of reproductive organs in female Japanese quail and on egg production were examined. Both oviducts and ovaries of retinoic acid-treated group developed more rapidly than those of both vitamin A-treated groups (experiment 1). Furthermore, retinoic acid-treated quail laid their first eggs earlier than the vitamin A-treated birds (experiments 1 and 2). These data suggest that retinoic acid accelerates development of the reproductive system in female Japanese quail. Recently, we reported that retinoic acid also accelerates testicular maturation in Japanese quail [17]. Taken together, retinoic acid appears to accelerate sexual maturation in both female and male Japanese quail.
Like oviduct and ovary weights, the mean body weight at 5 wk of age was also greater in RA-fed quail than in VA-fed quail, indicating that this rapid development of reproductive organs is accompanied by an increased body weight. As a consequence, the mean body weights at the first egg were not significantly different between the RA and VA treatment groups, even though the RA-treated quail began to produce eggs earlier (Table 2). Results of several studies indicate that the onset of egg laying in chickens and quail, which is synonymous with the onset of sexual maturity, depends on several biological factors, such as body weight [20, 21] and age [22, 23]. The onset of sexual maturity (i.e., age at the first egg laying), as indicated by the development of reproductive organs [24], requires a minimum body weight. Thus, the rapid increase of body weight in RA-fed quail may be the primary cause for the early onset of egg production, and it also may reflect changes related to early sexual maturity.
It could be argued that the early onset of sexual maturity might result in the laying of smaller eggs or in poor egg yield during the late experimental period. The observations that mean weights of the first egg were not different among the treatment groups, and that RA-fed quail laid more eggs (total weight and number of eggs per one bird; Table 2) than the VA-fed birds, excluded the possibility mentioned earlier. Taking these observations together, feeding retinoic acid diets to Japanese quail and, possibly, to chickens may help to improve the efficiency of their egg production.
Deficiency of vitamin A delays the onset of oviposition in chickens [4, 25] and quail [26]. In our experiments, quail receiving the vitamin A-supplemented diets (VA1 and VA2) began to lay eggs later than quail receiving the vitamin A-deficient diet supplemented with retinoic acid (Table 2 and Fig. 4). Vitamin A is extremely unstable and becomes rapidly oxidized. Thus, the possibility was considered that the vitamin A-supplemented diets used in our experiments (VA1 and VA2) were actually deficient in vitamin A. To prevent losses of vitamin A, we prepared all the diets every week and stored them in the dark at -20°C. In addition, the diets in the cages were renewed three times every day. The observation that vitamin A levels in the plasma and livers were replenished by the administration of both the vitamin A-supplemented diets (Fig. 1) ruled out any possible deficiency. We also found that eggs laid by these VA-treated birds had more than 90% hatchability when fertilized, and that those from quail receiving RA could not hatch (data not shown), confirming the existence of vitamin A in these vitamin A-supplemented diets. The incapability of the eggs from RA-fed hens to hatch resulted from the early embryonic death of these embryos [3, 27, 28]. Chen et al. [29] reported that retinoic acid, when provided as the only source of vitamin A to the adult quail, is not transferred to the egg. Thus, the early embryonic death may result from the complete vitamin A deficiency in these embryos [30]. These results suggest that the delay of oviposition in two VA-treated groups compared with the RA-treated group was caused by the vitamin A deficiency in the diets but, rather, that it was caused by an enhancing effect of retinoic acid on the development of reproductive organs. Thus, retinoic acid seems to have a more potent effect than vitamin A itself on development of the reproductive system in female Japanese quail. Many studies have examined the effects of retinoic acid in male mammals, but to our knowledge, few have examined such effects in females. Female rats given a vitamin A-deficient diet supplemented with retinoic acid can mate with normal males and conceive, but they frequently resorb their fetuses during pregnancy [2]. No attention has been paid on the effect of retinoic acid on sexual maturation in mammals.
The mechanism underlying the accelerative effects of retinoic acid on the sexual maturation of Japanese quail, however, is not well known. It is widely accepted that retinoic acid serves as the active form of vitamin A and alters gene expression at the level of transcription via its nuclear receptors. The expression of genes for RARs or RXRs as a function of vitamin A status has been examined in various tissues of adult animals, revealing tissue-specific downregulation of certain receptor isoforms by vitamin A deficiency and upregulation by repletion of vitamin A or retinoic acid [31–35]. Therefore, the effect of retinoic acid on the development of reproductive organs in Japanese quail may be an action of retinoic acid through its nuclear receptors to alter the expression of genes that relate to sexual maturation. Retinoic acid induces the expression of RARß mRNA in some mammalian tissues more quickly and strongly than retinol [10, 33, 35]. Follicle-stimulating hormone is a pituitary glycoprotein hormone essential for ontogeny and mature function of the gonads, and we recently found that expression of the FSH receptor and RARß genes in quail testis could be stimulated by retinoic acid (unpublished data). In addition, we have found that retinoic acid is easily taken up by reproductive organs of Japanese quail (data not shown), in contrast to the case with mammalian reproductive organs [36]. The exact mechanism underlying this action of retinoic acid, however, awaits further investigation.
In conclusion, the results presented here suggest that retinoic acid has a stimulating effect on the sexual maturation of female Japanese quail.
ACKNOWLEDGMENTS
We wish to thank Prof. Malden C. Nesheim, Prof. Tadashi Noguchi, and Dr. Derek LeRoith for their critical reading of the manuscript. We also wish to thank Teruo Ebihara for the gift of day-old female quail.
FOOTNOTES
1 Supported in part by a Grant-in-Aid for Exploratory Research (9876071) and by a Grant-in-Aid for the Japan Society for Promotion of Science Fellows to Z.F. (98218) from the Ministry of Education, Science, Sports and Culture of Japan. 
2 Correspondence: Hisanori Kato, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan. FAX: 81 3 5841 5114; akatoq{at}mail.ecc.u-tokyo.ac.jp
Accepted: July 31, 2000.
Received: April 11, 2000.
REFERENCES
, ß, and , and retinoid X receptors , ß, and , in the mouse testis. Endocrinology 1997; 138:1544–1551. mutant mice. Proc Natl Acad Sci USA 1993; 90:7225–7229. in the mouse. Cell 1993; 73:643–658.[CrossRef][Medline] in rat testis: in vivo response to depletion and repletion of vitamin A. Endocrinology 1998; 139:1239–1248., ß and retinoic receptor mRNA levels in various rat tissues. Biochem J 1992; 286:755–760.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
