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Testis |
INSERM U566-CEA-UNIVERSITE PARIS 7,3 CEA/DSV/DRR BP6, 92265 Fontenay aux Roses, France
INSERM U356,4 Centre de Recherche Biomédical des Cordeliers, 75270 Paris, France
INSERM U369 and IFR d'Endocrinologie,5 Faculté de Médecine Lyon-RTH Laennec, 69322 Lyon, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, Leydig cells, male sexual function, testis, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The differentiation and maintenance of the differentiated function of adult-type Leydig cells depend absolutely on LH (reviews in [9, 10]). In contrast, there is considerable evidence that differentiation of the fetal Leydig cells in rats does not require LH-like gonadotropins. Indeed, LH is not produced until the end of fetal development [11–14], and male gonads explanted on Fetal Day 12.5 and cultured for 3 days in medium without hormone spontaneously produce testosterone and respond to LH [15]. Furthermore, Leydig cell differentiation occurs in fetuses decapitated before the onset of LH secretion [12, 16]. In return, there is now considerable evidence indicating that many paracrine/autocrine factors are extremely important in regulating the development of fetal steroidogenic testicular function [3, 17, 18]. In previous studies, we focused on retinoic acid (RA), which is an important regulator of the development of many organs, including the lung and the kidney [19, 20]. Furthermore, RA and its precursor, vitamin A or retinol, are essential for the maintenance of normal reproductive function in adult male rats [21]. Vitamin A deficiency decreases plasma testosterone concentration [22] and retinoids increase steroid production by adult Leydig cells [23, 24]. Similarly, retinoids increase P-450 17
mRNA levels in cell lines derived from Leydig cells [25]. In contrast, we have shown that RA decreases the amount of testosterone produced in vitro by rat testis explanted on Fetal Day 14.5 [26].
In the present study, first we used our validated testicular organotypic culture system [7, 26–28] to investigate the ontogenesis and the characteristics of the effects of RA on testosterone secretion and to determine the molecular target of RA. We then investigated the physiological relevance of the involvement of RA in the regulation of fetal Leydig cell function using a rat model of mild vitamin A deficiency (VAD). Because total vitamin A deficiency induces severe malformations and does not allow normal pregnancy to proceed to completion, pregnant rats were exposed to partial vitamin A deficiency. This model allows overall normal fetal development as previously described [20].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Sprague-Dawley rats from Charles River (Saint Aubin Les Elbeuf, France) were housed in controlled photoperiod conditions (lights-on from 0600 to 0800 h) and fed commercial diets (U.A.R., Villemoisson sur Orge, France) with tap water ad libitum. Males were caged with females for the night. The day following an overnight mating was counted as 0.5 day postconception (dpc). Pregnant rats were anesthetized by intraperitoneal injection of 4 mg/ml sodium pentobarbital (Sanofi, Libourne, France) at various gestational ages, from 14.5 to 20.5 dpc. Testes were removed aseptically from male fetuses under a binocular microscope and immediately explanted in vitro. Natural birth occurs between Day 21.5 at 1300 h and Day 22.5 at 1800 h. Precise dating of postnatal development is important in our studies; only pups born between 1800 h on 21.5 dpc and 0800 h on 22.5 dpc (i.e., 70% of the neonates) were kept. Fetal Day 22.5 was counted as 0 day postpartum (dpp) for all newborns. The number of neonates in each litter was standardized at eight pups. Neonates were killed by cervical dislocation and their testes were immediately removed. All animal studies were conducted in accordance with the guidelines for Care and Use of Laboratory Animals from the French Ministere of Agriculture.
Chemicals and Solutions
The culture medium used was Ham F12/Dulbecco modified Eagle medium (1:1) (Gibco, Grand Island, NY) containing 0.35% glutamine (Flow Laboratories, McLean, VA) and 80 µg/ml gentamicin (Gentalline, Schering-Plough, Levallois-Perret, France). Ovine (o) LH (NIH.LH S19; 1.01 NIH.LH.S1 units/mg) was a gift from Dr. A.F. Parlow (NIDDK, Bethesda, MD) and recombinant human (h) LH (14 610 IU/mg) was provided by Serono, Levallois-Perret, France. All-trans retinol (RE) and all-trans retinoic acid (RA) were purchased from Sigma (Saint Quentin-Fallavier, France). Anti-3ß-hydroxysteroid dehydrogenase (3ßHSD) antibody was generously provided by Dr. G. Defaye (INSERM U244; Grenoble, France).
Organ Cultures
Testes were cultured on Millipore filters (pore size, 0.45 µm) as previously described [7]. Briefly, intact 14.5-dpc and 15.5-dpc testes were placed on filters. Testes from older animals were cut into small pieces (2, 4, 8, 16 pieces respectively for 16.5, 18.5, 20.5 dpc and 3 dpp) and all the pieces from the same testis were placed on a single Millipore filter. The filter bearing the pieces of testis was floated on 0.4 ml (14.5-dpc testes) or 1.5 ml (testes from older animals) of culture medium in tissue culture dishes and cultured at 37°C, in a humidified atmosphere containing 95% air:5% CO2 for 3 days. The medium was changed every 24 h. The response to retinoids was measured by comparing one testis cultured in medium containing retinoic acid or retinol with the other testis from the same fetus cultured in medium without retinoids, which served as the control.
In some experiments, at the end of the culture period, we added 100 ng/ml oLH or 0.5 IU/ml recombinant hLH to all media for the last 3 h of culture. For cellular analyses, the whole explant was fixed for 2 h at 4°C in Bouin fluid, embedded in paraffin, and 5-µm sections were cut.
Identification and Counting of Leydig Cells
Leydig cells were identified by immunocytochemical detection of 3ß- hydroxysteroid dehydrogenase (3ßHSD), as previously described [26]. Immunostaining was performed with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Deparaffinized sections were rehydrated, placed in a container with citrate buffer, and heated in a microwave oven at 650 W for 5 min, and then the container was refilled with fresh buffer. This operation was repeated three times. Endogenous peroxidases and nonspecific protein binding were blocked by incubation in hydrogen peroxide followed by normal goat serum. The sections were then incubated overnight with anti-3ßHSD antibody (1:400) in a humidified chamber at 4°C. Binding of this primary antibody was detected by incubation with a biotinylated goat anti-rabbit secondary antibody and the avidin-biotin-peroxidase complex. Peroxidase activity was detected with 3,3'-diaminobenzidine. Sections were rinsed in PBS between each step. The specificity of 3ßHSD staining was checked by replacing the antibody with nonimmune IgG.
We counted all the Leydig cells on every third section of whole testis and applied the Abercrombie formula, as previously described [26].
Testosterone Radioimmunoassay
The testosterone secreted into the medium was measured in duplicate by radioimmunoassay as previously described [5]. No extraction or chromatography was performed because 17ß-hydroxy-5-androstan-3-one, the only steroid that displays significant cross-reaction (64%) with testosterone, is secreted only in tiny amounts by the fetal rat testis [5].
Cyclic AMP Radioimmunoassay
Cyclic AMP production was assessed after 72 h of culture by incubating the testes for 3 h with fresh medium containing 1 mM IBMX in the presence or absence of 0.5 IU/ml recombinant hLH. The media were collected, acetylated, and assayed for cAMP by RIA [29].
Reverse-Transcriptase Polymerase Chain Reaction
Gene expression in fetal testes was evaluated by reverse transcriptase- polymerase chain reaction (RT-PCR). At the end of the culture period, total RNA was extracted from fetal or neonatal testes, with the RNA Plus kit (Bioprobe Systems, Montreuil-sous-Bois, France) according to the manufacturer's instructions. The expression of P450 cholesterol side-chain cleavage (P450scc), 3ßHSD, P450 17 -hydroxylase/C17-20 lyase (P450C17), steroidogenic acute regulatory protein (StAR), and ß actin was studied by RT-PCR with the following specific primers: for StAR (sense [S]), 5'-ACAACCAGGAAGGCTGGAG-1;3' and (antisense [AS]), 5'-ATGCAGGTGGGACCGTGTTCA-1;3'; for P450scc (S), 5'-AGGTCTTTGCCTGCGCT-1;3', and (AS), 5'-GCATCTCTGTGATGTTGG-3'; for P450C17 (S), 5'-GCCTGACGGACATTCTG-3', and (AS), 5'-TCGTGATGCAGTGCCCAG-3'; for 3ßHSD (S), 5'-TGGTGACTGGAGCAGGA-3', and (AS), 5'-AAGAAGCTCACAATTTCCAGC-3'; for ß actin (S), 5'- AAGAGAGGCATCCTGACCCT-3', and (AS), 5'-GGCCATCTCTTGCTCGAAGT-3', with respective predicted products size of 380, 950, 420, 890, and 500 base pairs [30]. The intensity of the bands obtained was determined by densitometry with image analysis software (NIH Image 1.62 software for Macintosh).
In Vivo Experiments
Twenty-four-day-old female Sprague-Dawley rats (just after weaning) were assigned to three groups. The control group received the standard diet (A 03, containing 16 800 IU/kg vitamin A). Animals of the vitamin A-deficiency group (VAD) were fed the same diet but with no vitamin A or C (100 ± 100 IU/kg). The third group (VAD + vitamin A) received the deficiency diet supplemented exclusively with vitamin A (+ 16 800 IU/kg vitamin A). The standard and vitamin A-deficient diets were purchased from UAR Laboratory (Villemoison/Orge, France). Blood samples were taken regularly from the tail and plasma was saved for retinol measurement. Blood was drawn once a week during the first 2 mo of the diet and twice a week afterward. In order to induce a mild vitamin A deficiency in fetuses and neonates, females were caged with the males as soon as retinol concentration began to fall. This usually happens between 6 and 9 wk from the beginning of the diet. It is of particular importance to perform the mating very early while the VAD is still partial, as severe VAD is not compatible with a successful pregnancy and would trigger abnormalities and major growth retardation in the offspring. On the morning after mating, vaginal smears were performed to detect the presence of sperm. The day of the detection of the sperm was considered as Fetal Day 0.5 at noon. Diets remained unchanged until the term of the pregnancy or until an animal was killed and during lactation.
Plasma Retinol Determination
Plasma vitamin A concentration was determined by reversed phase HPLC [31]. Briefly, vitamin A was extracted from plasma samples with ethanol/n-hexane/butylated hydroxytoluene (33%:67%:5%), retrieved in the hexane phase, dried by evaporation under nitrogen. The residue was dissolved in 200 µl of the mobile phase (methanol:dichloromethane, 65: 35), and 150 µl of this solution was injected into the HPLC pump (Waters, Saint-Quentin en Yvelines, France), linked to a multiwavelength detector. Detection was performed at 325 nm, at a flow rate of 2 ml/min on a Nucleosil C-18 column with a precolumn module (Life Science International, Cergy Pontoise, France). Retinol was used as the external standard and retinol palmitate as the internal standard. All reagents were of ultrapure grade (Sigma).
Statistical Analysis
All values are expressed as means ± SEM. The significance of differences between mean values for the treated and untreated controls was evaluated using Student paired t-test. Other means were compared by one- way ANOVA (with Fisher test).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ontogenesis of the effect of RA on basal and acute LH- stimulated testosterone secretion in the rat fetal and neonatal testis Testes were removed from fetuses on Days 14.5, 16.5, 18.5, and 20.5 postconception and from neonates on Day 3 after birth. They were cultured for 3 days in the absence of LH, with or without 10–6 M RA (Fig. 1). At the end of the culture, in order to assess the steroidogenic capacity of Leydig cells, we performed an acute stimulation by LH. For testes explanted before 18.5 dpc, testosterone levels in control medium increased during the culture period in a pattern mimicking that observed in vivo [4, 5], and the steroidogenic response to acute stimulation by LH was very strong. For testes explanted after 18.5 dpc, lower levels of testosterone secretion were observed and the steroidogenic response to acute stimulation by LH was weaker than that observed in younger fetuses. Retinoic acid reduced both basal and acute LH-stimulated testosterone secretion by 14.5 and 16.5-dpc testes, but had no effect on testes removed from 18.5 dpc onward.
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Effect of retinol and various concentrations of retinoic acid on fetal Leydig cell function and number Testes from 16.5-dpc fetuses were cultured for 75 h with or without various concentrations of RE or RA (Fig. 2). RA decreased both basal and acute LH-stimulated testosterone secretion in a dose-dependent manner. The lowest effective dose was 3 x 10–8 M RA. Like RA, RE decreased both basal and acute LH-stimulated testosterone secretion. However, RE was less potent than RA. The negative effect of 10–6 M RE was not detected until the third day in culture, whereas RA at the same concentration acted more rapidly. Last, 10–6 M RE inhibited basal and LH-stimulated testosterone secretion on Day 3 but significantly less strongly than did 10–6 M RA. Nevertheless, these results suggest that fetal testis is able to convert RE into bioactive RA.
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Before and after 3 days of culture in the presence or absence of 10–6 M RA, we counted Leydig cells, identified by immunohistochemical detection of 3ßHSD (Fig. 3). In control medium, the number of 3ßHSD-positive cells increased during culture, suggesting that the cellular differentiation of new fetal Leydig cells occurs spontaneously in vitro. RA did not affect the number of Leydig cells.
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Effects of RA on basal and acute LH-stimulated cAMP production In order to investigate the level of the negative effects of RA, we assess the effects of a 3-day RA treatment on basal and acute LH-stimulated cAMP secretion in cultured 16.5-dpc testes. We chose to study testes at this stage rather than 14.5-dpc testes because the low cAMP of the latest is hardly quantifiable. We used recombinant LH to prevent the stimulation of cAMP production in Sertoli cells by the small amounts of FSH that are suspected to contaminate oLH preparations. RA decreased acute LH-stimulated cAMP production but had no effect on basal secretion (Fig. 4).
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Effects of RA on steroidogenic enzyme mRNA levels We analyzed the effect of RA on mRNA levels for P450scc, 3ßHSD, P450C17, and StAR by RT-PCR. Testes removed from 16.5-dpc and 3-dpp animals were used because testes at these stages display RA-inhibited and RA-insensitive testosterone secretion, respectively. For 16.5-dpc testes, only the level of P450C17 mRNA was significantly decreased by 3 days of treatment with RA (Fig. 5A). With 3-dpp testes, RA decreased mRNA levels for all steroidogenic enzymes, expressed with respect to levels of ß actin mRNA. However, we have previously shown that, at this stage (but not 16.5 dpc), RA increases the number of Sertoli cells by a factor of 1.3 [26]. Sertoli cells are the most abundant cell type in the testis (90% of total testicular cells at 3 dpp). Thus, the ß actin mRNA levels obtained after RA treatment must be 1.3 x (90/100) times higher than those of the controls. To take into account this increase, the values of Leydig cell markers must be multiplied by (1.3 x 0.9). In Figure 5, the dashed lines represent these corrected values. After this correction, we found that RA also reduced P450C17 mRNA levels in 3-dpp testes without changing the levels of mRNA for other enzymes, as observed with 16.5-dpc testes (Fig. 5A).
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Changes in steroidogenic enzyme-relative expressions in cultures To better understand the differences in RA responses between the fetal (16.5 dpc) and the neonatal testis (3 dpp), we measured the expression of each steroidogenic enzyme and reported it to the sum of the expression of the four enzymes after 3 days of culture in basal medium (Fig. 5B). Although no conclusion can be drawn for a single age, as the relative expression at one given stage may appear as the result of the PCR conditions, it clearly appears that two changes occurred in the relative importance of the steroidogenic enzymes. The P450C17-relative expression increased while the one of the 3ßHSD decreased between 16.5-dpc and 3-dpp testes. In light of the previous effect of RA on P450C17, it is important to note that P450C17-relative expression almost doubled during the studied period (from 23% ± 2 % in 16.5-dpc testes to 43% ± 10% in 3-dpp testes).
In Vivo Experiments
To investigate the physiological relevance of the involvement of RA in the regulation of fetal steroidogenesis and to determine whether RA exerted a positive or negative effect in vivo, we used a mild vitamin A deficiency model previously validated in the rat [20].
Effect of vitamin A deficiency on plasma retinol concentration and growth From weaning (24 dpp) onward, female rats were fed one of three diets: 1) a standard diet (control), 2) a vitamin A-deficient diet (VAD), and 3) the vitamin A-deficient diet supplemented with vitamin A (VAD + Vit A). Regardless of the diet of the growing rats, plasma retinol concentrations decreased for about 1 month and then stabilized during the second month (Fig. 6). In the VAD group, plasma retinol concentration continued to fall after 60 days on the diet and remained low after 75 days on the diet. The increase in body weight of the rat during the diet period was not affected by the type of diet (data not shown). Females of the VAD group were mated with males during the period of specific decrease in plasma retinol concentration in the VAD group (60–72 days of diet). VAD diet modified neither the weight of placenta nor the weight of fetuses at 15.5 and 18.5 dpc and of 3-day neonates (data not shown).
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Effect of VAD on the steroidogenic activity and differentiation of fetal and neonatal testes The steroidogenic activity and differentiation of the fetal testis in vivo can be evaluated by measuring the production of testosterone during a short incubation ex vivo in the absence (basal) and presence of LH, respectively [12, 16]. For the animals on the control diet, the ex vivo basal testosterone secretion increases about three times between 15.5 and 18.5 dpc and then sharply decreases to reach a level six times lower on 3 dpp. These kinetics fit well with what has already been described [3, 5, 6, 8]. The stimulation in response to LH of testosterone secretion as a percentage of the basal value is much stronger on 18.5 dpc than on 15.5 dpc; this was also previously reported. Basal and LH-stimulated testosterone secretions of the testes ex vivo were not affected by VAD on Fetal Day 15.5, but were slightly higher on Fetal Day 18.5; and on Neonatal Day 3, basal testosterone secretion of VAD animals was five times higher than the one of animals on the control diet (Fig. 7).
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Interestingly, the basal testosterone secretion of the VAD neonate testes ex vivo was much higher than the one for the control animals, whereas no negative effect of RA was observed in vitro from 18.5 dpc onward. In order to understand this point, we compared the changes observed in vitro in testes from VAD fetuses with those in testes from VAD supplemented with vitamin A fetuses (Fig. 8). VAD did not affect basal and LH-stimulated testosterone secretion in 15.5-dpc testes cultured in control medium for 3 days. In these cultures, the addition of RA similarly inhibits the testosterone secretion from testes from VAD and testes from VAD + vitamin A fetuses. With 18.5-dpc VAD fetuses, in control medium, basal levels of testosterone secretion were maintained during the 3 days of culture whereas testosterone secretion decreased in cultured testes from VAD + vitamin A fetuses, as it did in the control (Fig. 1). The addition of RA in the medium reduced the ability of 18.5-dpc VAD testes to maintain basal levels of testosterone secretion. Furthermore, the acute LH response of the testes doubled in VAD testes after 3 days of culture in the absence of RA; but when the testes were cultured in the presence of RA, no significant change could be detected between VAD + VitA and VAD animals. In conclusion, vitamin A deficiency in vivo, followed by retinoid deprivation in vitro, prevents the decline in testosterone secretion observed in rats after 18.5 dpc. This effect can be partially reversed by in vitro supplementation with RA. These observations are consistent with the large increase in ex vivo testosterone secretion of 3-dpp VAD neonates shown in Fig 7.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We investigated various enzymes involved in steroidogenesis and found that only P450C17 mRNA levels were reduced by RA in vitro. This enzyme seems to be the most sensitive steroidogenic enzyme in rat fetal testis. Indeed, TGFß1 inhibited testosterone production by specifically decreasing P450C17 mRNA levels in rat fetal testicular cells [34] and in pig Leydig cells [35]. Curiously, it has previously been reported that RA increases P450C17 mRNA levels in a cell line derived from Leydig cells [25]. This apparent discrepancy with our result may be due to the difference in the culture systems, as Leydig cells do not respond the same way in a dispersed cell culture or in organ culture [34], the latest system displaying kinetics closer to the ones observed in vivo. It is noteworthy to point out that this may also reflect the fact that specific regulations exist in the fetal-type Leydig cells. Surprisingly, RA decreased P450C17 mRNA levels in both 16.5-dpc and 3-dpp cultured testes, whereas it reduced testosterone secretion only in 16.5-dpc testes. This observation led us to hypothesize that this enzymatic step may be rate limiting on 16.5 dpc but not on 3 dpp, and, therefore, RA would cease to inhibit testosterone secretion after 18.5 dpc probably because its final target (P450C17) ceases to be rate limiting. This hypothesis was strengthened by our observation that the relative expression of this enzyme compared with the expression of the other steroidogenic enzymes increases markedly between 16.5-dpc and 3-dpp cultured testes.
Our results showed that RA diminished the cAMP production increase in response to LH. Therefore, in vitro, RA may decrease LH-stimulated testosterone secretion at least partially by inhibiting cAMP production. RA also induced a decrease in FSH-induced cAMP production by Sertoli cells in the fetal testis [26]. Thus, RA seems to have similar effects on gonadotropin-stimulated cAMP production in all somatic cells of rat fetal testis, suggesting it acts on a common pathway.
Because responses were obtained in vitro with 10–6 M retinol, which is in the range of circulating concentrations observed in fetuses [20], we investigated furthermore the physiological relevance of the effect of RA on fetal testicular steroidogenesis. We achieved a moderate decrease in circulating vitamin A levels, which didn't affect fetal growth. This resulted in a threefold decrease in maternal plasma vitamin A concentration at term. A similar decrease must also occur in the fetal blood compartment because plasma retinol concentrations are correlated in mother and fetus [20].
The major finding of this study is the demonstration, for the first time, that retinol can regulate fetal and neonatal testicular steroidogenesis in vivo. Mild VAD results in an increase in basal and LH-stimulated testosterone secretion by the testes during late fetal and neonatal life. As Leydig cells start producing testosterone at 15 dpc in the rat [5, 36], we expected VAD to have no consequence at 15.5 dpc. This observation was nonetheless important to perform, as severe VAD can trigger fetal malformations [37]. It demonstrates that, in our model, vitamin A deficiency was mild enough not to disturb normal initial development of the testis.
VAD resulted in an increase in testicular steroidogenesis in neonates whereas in vitro RA had no effect at this developmental stage. However, the steroidogenic activity of the testes from VAD 18.5-day fetuses cultured for 3 days in the absence of RA did not decrease as a function of the time, whereas it did following the addition of RA to the medium. Thus, long-term vitamin A deficiency may result in an inhibition of the functional regression of Leydig cells (i.e., the decrease in testosterone production) observed in vivo during late fetal life.
It is important to note that the inhibitory effect of retinoids on fetal and neonatal Leydig cell activity and differentiation reported here are specific to the fetal Leydig cell type. Indeed, in the adult rat, VAD has the opposite effect, causing a decrease in plasma testosterone concentration [22].
Taken together, our results evidenced two major effects of RA. On the one hand, before 18.5 dpc, RA is able to decrease fetal Leydig cell function and/or differentiation in vitro, and the reduction of retinol level in vivo results in an increase of the steroidogenic testicular function. On the other hand, after 18.5 dpc, RA has no more effect in vitro, but the maintenance of a normal level of retinol in vivo is required for the normal regression of fetal Leydig cell function that occurs during this period.
In conclusion, our in vivo study demonstrated, for the first time, the involvement of retinoids in the control of steroidogenic activity in the fetal and neonatal testis. Thus, the inhibitory effect of RA observed in vitro is physiologically relevant. We now need to determine whether this effect is specific to rats or is also observed in humans.
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FOOTNOTES |
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Received: 25 July 2003.
First decision: 18 August 2003.
Accepted: 3 February 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5-4-isomerase and 17-hydroxylase/C17-20 lyase cytochrome P-450 messenger ribonucleic acid levels in the K9 mouse Leydig cell line. Mol Cell Endocrinol 1994 106:31-39[CrossRef][Medline]This article has been cited by other articles:
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  R. Lambrot, H. Coffigny, C. Pairault, A.-C. Donnadieu, R. Frydman, R. Habert, and V. Rouiller-Fabre Use of Organ Culture to Study the Human Fetal Testis Development: Effect of Retinoic Acid J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2696 - 2703. [Abstract] [Full Text] [PDF]
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