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Molecular Characterization of Postnatal Development of Testicular Steroidogenesis in Luteinizing Hormone Receptor Knockout Mice

Department of Physiology, Institute of Biomedicine, University of Turku (F.P.Z., T.P., F.Z., M.P., I.H.), Fin-20520 Turku, Finland; Biomedicum Helsinki, Institute of Biomedicine/Physiology, University of Helsinki (F.P.Z.), 00014 Helsinki, Finland; and Institute of Reproductive and Developmental Biology, Imperial College London (I.H.), London, United Kingdom W12 0NN

Address all correspondence and requests for reprints to: Ilpo Huhtaniemi, M.D., Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, Du Cane Road, London, United Kingdom W12 0NN. E-mail: ilpo.huhtaniemi{at}imperial.ac.uk.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We recently demonstrated that the sexual development of LH receptor (LHR) knockout mice is normal until birth, but is totally arrested thereafter. To study further the functional defects of LHR knockout mice, the expression of selected Leydig cell-specific genes was studied in (-/-) and control (+/+) mice between birth and adulthood. Testis weights were similar at birth in both types of mice, but after about 3 wk, the (-/-) testes remained significantly lighter, weighing only 18% of (+/+) testes on d 70. Testicular testosterone (T) content on d 1 was also similar in (-/-) and (+/+) testes, but it was 97% reduced by d 70 in the former. Likewise, testicular T production in vitro was similar in neonatal (-/-) and (+/+) testes, but became undetectable in adult (-/-) testes. The mRNA expression of cytochrome P450 side-chain cleavage, 17-hydroxylase cytochrome P450, 17ß-hydroxysteroid dehydrogenase type III, 3ß-hydroxysteroid dehydrogenase I (3ßHSDI), steroidogenic acute regulatory protein, and relaxin-like factor were similar in newborn (-/-) and (+/+) testes, but became gradually very low/undetectable in (-/-) mice. The only exception was the persistently high expression of 3ßHSDI in peritubular Leydig precursor and mesenchymal cells of the (-/-) testes at all ages. Immunohistochemistry and Western hybridization studies confirmed the above findings. In conclusion, LH action is not essential for the differentiation and function of mouse fetal Leydig cells, but, with the exception of 3ßHSDI, the expression of the key genes of endocrine function of adult Leydig cells is dependent on LH signaling.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE TWO GONADOTROPIN receptors, LH receptor (LHR) and FSH receptor (FSHR), play a crucial role in mediating the action of gonadotropins on reproductive functions in their target cells in the testis and ovary. LHR is expressed in the testis mainly in Leydig cells, and upon binding of LH, it stimulates their steroidogenesis. There are two growth phases of Leydig cells during testicular development in the mouse and most other mammalian species (1, 2). The first type, i.e. fetal Leydig cells, originate prenatally, shortly after testicular differentiation in utero, and they produce the androgen required for fetal masculinization as well as relaxin-like factor (RLF; also called insulin-like factor 3) that regulates the trans-abdominal phase of testicular descent. The second generation of adult Leydig cells appears postnatally along with pubertal development (2, 3, 4). The adult Leydig cells originate from undifferentiated fibroblast-like cells or mesenchymal cells in the testicular interstitium (5, 6). The fate of fetal Leydig cells after puberty has been debated for years (7), although recent studies of the Dhh-null mouse support the hypothesis that the cells remain in the testis, but are eclipsed in size and number by the developing adult-type Leydig cells (8). A body of evidence indicates that although fetal Leydig cells express LHR and are responsive to LH stimulation, they are capable of producing sufficient levels of androgens in the absence of LH stimulation to induce male fetal masculinization (2, 9, 10, 11, 12). Likewise, indirect evidence from several studies suggests that LH is not required at the onset of adult Leydig cell differentiation, although it is essential for later stages in the Leydig cell lineage (13, 14).

We have recently generated the LHR knockout (LuRKO) mouse by inactivating through homologous recombination exon 11 on the LHR gene (15). LuRKO males and females were born phenotypically normal, but postnatally their testicular growth and descent as well as external genital and accessory sex organ maturation were blocked. To study further the molecular defects related to LHR inactivation and to better understand the role of LHR signaling in Leydig cell development and steroidogenesis at the different stages of development, the expression of several important enzymes and proteins related to Leydig cell endocrine function were studied in LuRKO testes between birth and adulthood.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of LuRKO mice
The strategy used for targeted disruption of the LHR gene and a preliminary description of the phenotype have been presented previously (15). Briefly, exon 11 of the mouse LHR gene was replaced by the neo gene. Targeted disruption of exon 11 of the LHR gene resulted in complete loss of LHR binding in both intact cells and detergent-solubilized extracts and in consequent loss of LHR function. The heterozygous founder mice of the 129/SvEv/C57BL/6J strain were bred with the C57BL/6J strain, and mice of F₂ and F₃ generations of these crossings were used in the present study. The mice were genotyped by PCR on tail DNA as previously described (15). All animal procedures were approved and performed in accordance with the institutional animal care police of the University of Turku. For postnatal ages, the day of birth was designated d 1 of life.

Tissue collection and histology
Wild-type (WT; +/+) and LuRKO (-/-) mice were obtained from the same colony at different ages. Animals were anesthetized by avertin. Testes were removed and weighed, then fixed in 4% paraformaldehyde at 4 C for 12 h, dehydrated, and embedded in paraffin, and 5-µm-thick sections were prepared. Sections were stained with Harris’ hematoxylin and eosin (BDH Ltd., Poole, UK). The reproducibility of all of the morphological data was verified by similar findings in at least three different animals.

RNA isolation and semiquantitative RT-PCR
Total RNA was isolated from the testicular samples using a single-step extraction method as described previously (16). The quality of extracted RNA was determined by spectrophotometry and by electrophoresis of RNA on formaldehyde-agarose gel. RNA samples with an A26/A28 ratio greater than 1.8 and distinct 28S and 18S bands were used for further study.

The patterns of mRNA expression of cytochrome P450 side-chain cleavage (P450scc), 17-hydroxylase cytochrome P450 (P450-17OH), 3ß-hydroxysteroid dehydrogenase type I (3ßHSDI), 17ß-hydroxysteroid dehydrogenase type III (17ßHSDIII), steroidogenic acute regulatory protein (StAR), RLF, and ß-actin in the testes were assessed at the different ages by semiquantitative RT-PCR using specific primer pairs, as listed in Table 1. Total RNA was isolated from testicular samples at different age as described above. For amplification of the specific target genes, RT and PCR reactions were run in separate steps. In brief, equal amounts of total testicular RNA (4 µg) were heat-denatured and reverse transcribed by incubating at 42 C for 1 h and at 52 C for another 1 h with 12.5 U avian myeloblastosis virus reverse transcriptase (AMV-RT; Promega Corp., Madison, WI), 20 U ribonuclease inhibitor (RNasin, Promega Corp.), 200 nM deoxy-NTP mixture, and 1 nM random hexamers (Promega Corp.) in a final volume of 30 µl of 1x AMV-RT buffer. The reactions were terminated by heating at 97 C for 5 min and cooling on ice, followed by diluting the samples with nuclease-free H₂O (final volume, 100 µl). Liquid controls and RT reactions without AMV-RT were run in parallel with the testicular samples. For semiquantitative PCR, 10-µl aliquots of the cDNA samples were amplified in 50 µl 1x PCR buffer in the presence of 2.5 U Taq DNA polymerase (Promega Corp.), 200 nM deoxy-NTP mixture including [-³²P]CTP, and the appropriate primer pairs (1 nM of each primer; see Table 1). PCR reactions consisted of a denaturing cycle at 97 C for 5 min, followed by a variable number of cycles of amplification defined by denaturation at 96 C for 1 min, annealing at 55–62 C for 1 min, and extension at 72 C for 2 min. A final extension cycle at 72 C for 15 min was included. The number of cycles was optimized to ensure amplification in the exponential phase of PCR. Different numbers of cycles were tested for different specific genes. Based on the analysis of cycle dependency of the intensity of the PCR signals generated, 37 cycles for P450-SCC, P450-17OH, and StAR; 31 cycles for 3ßHSDI; 38 cycles for 17ßHSDIII; 35 cycles for RLF; and 20 cycles for ß-actin were used.

Real-time RT-PCR
The fetal Leydig cell marker, thrombospondin (TSP2), and the adult Leydig cell marker, 3ßHSDVI, were analyzed by real-time RT-PCR with specific primer pairs (Table 1) during testicular development. RNA was isolated as described above. Real-time RT-PCR analysis was performed using the DNA Engine Opticon system (MJ Research, Inc., Waltham, MA) with continuous fluorescence detection. Briefly, 2 µg RNA were treated with deoxyribonuclease (Invitrogen, Carlsbad, CA). PCR reaction was performed using the QuantiTect SYRB Green RT-PCR Kit (Qiagen, Valencia, CA). One hundred nanograms of RNA were used in each reaction. All samples and standards were run in triplicate following the program for 30 min at 50 C (RT reaction) and for 15 min at 95 C (denaturation), followed by 30 PCR cycles for 15 sec at 94 C and for 30 sec at 54 C (TSP2 and ß-actin) or at 59 C (3ßHSDVI) or for 30 sec at 72 C. TSP2 and 3ßHSDVI levels were analyzed in proportion to the ß-actin level. ß-Actin was used as an endogenous control to equalize unequal amounts of RNA.

Northern hybridization analysis
For Northern hybridization analyses, RNA samples (15 µg/ml) were resolved onto 1.2% denaturing agarose gels and transferred onto Hybond-XL nylon membranes (Amersham International, Aylesbury, UK) using the capillary method. The membranes were cross-linked by short-wave UV irradiation and prehybridized for at least 4 h at 65 C in a solution containing 50% deionized formamide, 3x standard saline citrate (SSC), 5x Denhardt’s solution, 0.1 g/liter heat-denatured calf thymus DNA, 1% sodium dodecyl sulfate (SDS), and 0.1 g/liter yeast transfer RNA. Hybridization was carried out at 66 C (cRNA probes) or 42 C (cDNA probes) overnight in the same prehybridization solution after adding the corresponding radiolabeled probes. After hybridization, the membranes were washed in 2x SSC/0.1% SDS at room temperature for 20 min, in 0.5x SSC/0.1% SDS twice for 30 min each time at 65 C, and twice in 0.1x SSC/0.1% SDS for 1 h at 65 C. The filters were exposed to Kodak x-ray films (Kodak XAR-5, Eastman Kodak Co.) at -70 C for 24–72 h.

For hybridization, ³²P-labeled cRNA and cDNA probes specific for the target genes were generated using the Riboprobe system II kit and the Prime-a-Gene Labeling kit (Promega Corp.), respectively. For generation of cRNA probes, DNA fragments generated by RT-PCR, corresponding to 186–673 of P450scc cDNA, 55–616 of P450-17OH cDNA, 175–609 of 17ßHSDIII cDNA, and 207–703 of StAR cDNAs, were subcloned into T-vector (Promega Corp.) and used as templates. For cDNA probes, DNA fragments generated by RT-PCR, corresponding to 156–556 of 3ßHSDI and 21–299 of RLF cDNAs, were used as templates (Table 1).

Immunohistochemistry
Testicular sections (5 µm thick) were cut from each sample. After rehydration, the slides were washed twice in Tris-buffered saline (TBS; 10 mM Tris-HCl, pH 8.0, and 100 mM NaCl) for 5 min each time. For P450scc and P450-17OH staining, all of the sections were treated for antigen retrieval by microwaving at 700 watts for 15 min in 10 mM sodium citrate solution, pH 6.0. After two washes with TBS, 50 µl blocking solution (TBS containing 1% BSA and 3% normal horse serum) were applied on each section and incubated for 1 h at room temperature. After blocking, 100 µl primary antibody diluted (P450scc, 1:1000; P450-17OH, 1:1200; 3ßHSDI, 1:600) in TBS, containing 1% BSA or normal rabbit serum, were applied to each slide and incubated at 4 C overnight. On the following day, the slides were washed in PBS and incubated with second antibody, and positive cells were visualized using the Vectastain Elite kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. Antibodies against 3ßHSDI, P450scc, and P450-17OH were donated by Drs. J. I. Mason (University of Edinburgh, Edinburgh, UK), A. H. Payne (Stanford University, Palo Alto, CA), and M. R. Waterman (Vanderbilt University, Nashville, TN).

Western blot
Testes from different age groups were lysed in a buffer containing 25 mM Tris-HCl, 120 mM NaCl, 0.5% Nonidet P-40, 4 mM NaF, 100 µM Na₃VO₄, 100 IU/ml aprotinin, 1 mM phenylmethylsulfonylfluoride, and 10 mg/liter leupeptin at 4 C for 30 min with vigorous shaking. Cell lysates were centrifuged for 20 min at 13,000 x rpm, and the supernatants were transferred to new tubes. The concentration of protein was measured by the Bradford method. One hundred micrograms of protein from the different age groups were diluted (4:1) in sampling buffer (100 mM Tris-HCl, 20% glycerol, 2% SDS, 0.1 M dithiothreitol, and 0.01% bromophenol blue) and boiled for 3 min. The proteins were resolved on 12.5% SDS-polyacrylamide gels using the Mini Protean II system (Bio-Rad Laboratories, Hercules, CA). After electrophoresis, the proteins were electrophoretically transferred onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ).

The membranes were incubated in blocking buffer [10 mM Tris-HCl (pH 8.0), 0.1% Tween 20, and 5% nonfat milk powder] at room temperature for at least 1 h, followed by incubation in blocking buffer containing the primary antibody for 1 h. After three washes with 10 mM Tris-HCl (pH 8.0) and 0.1% Tween 20, the membranes were incubated in blocking buffer containing the secondary antibody for 1 h. After three washes, the membranes were subjected to chemiluminescent detection using the ECL Western Blotting Detection Kit (Amersham Biosciences), and finally the membranes were exposed to Fuji x-ray film (Fuji Photo Film Co. Ltd., Tokyo, Japan) for 1–10 min.

Forskolin stimulation test
Testicular samples were obtained from 1- and 70-d-old (+/+) and (-/-) mice. The testes were removed immediately after decapitation, decapsulated, and cut into pieces of approximately equal size. The testis slices were then incubated in 2 ml DMEM/Ham’s F-12 medium (1:1; Life Technologies, Inc., Paisley, UK), supplemented with 10% fetal calf serum and 0.1 g/liter gentamycin (Biological Industries, Bet-Hemeek, Israel) in a shaking water bath at 32 C under an atmosphere of 5% CO₂-95% O₂. After a 30-min preincubation, the medium was replaced by fresh medium (DMEM/Ham’s F-12) with or without 5 µmol/liter forskolin (Sigma-Aldrich Corp., St. Louis, MO). The optimal stimulation time was 4 h. At the end of this period, the incubation media were collected, and testosterone (T) was measured from a 100-µl sample (see below). The testicular samples were collected and weighed, and the level of T in the medium was expressed as normalized values per gram of incubated tissue.

Hormone measurements
Intratesticular T was determined by homogenizing (-/-) and (+/+) testes from 1- and 70-d-old mice. For 1-d-old (+/+) and (-/-) mice and 70-d-old (-/-) mice, whole testes were used, whereas for 70-d-old (+/+) mice only a proportion of testes was homogenized.

For measuring the T concentration, 100 µl testicular homogenate or culture medium were extracted twice with 2 ml diethyl ether and evaporated to dryness overnight in a fume hood. After reconstitution into PBS, T was measured by a standard RIA (17). Protein concentrations in homogenates were determined using the Bradford method (18).

Densitometry
The relative ODs of mRNA, semiquantitative RT-PCR products, and 18S ribosomal RNAs levels were quantified using the TINA 2.0 program (Raytest, Straubenhardt, Germany).

Statistical analysis
The StatView program (Windows version 4.57, Abacus Concepts, Inc., Berkeley, CA) was used for ANOVA and t tests. Significance was set at P < 0.05. The values are presented as the mean ± SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Testicular phenotype of LuRKO mice
The age-related increase in the postnatal testis weights of (+/+) and (-/-) mice are shown in Fig. 1. Testis weights on d 1, 5, and 10 were nearly the same in both groups of mice, but from d 20 onward, the growth rate of (-/-) testes was much slower. On d 70, they only weighed about 18% of the (+/+) testes.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Testis weights during postnatal development of (+/+) and (-/-) mice. The values are the mean ± SEM of at least 10 animals. ***, P < 0.001 compared with age-matched (+/+) groups.

View larger version (166K): [in this window] [in a new window]   FIG. 2. Representative of photomicrographs of testicular tissue from the mice of different ages. A–E and A'–E', Testicular histology from different ages of (+/+) mice. F–J andF'–J', Testicular histology from different ages of (-/-) mice. A'–E' and F'–J', as in A–E and F–J, but at higher magnification. Representative fetal Leydig cells (FLC), adult Leydig cells (ALC), peritubular myoid cells (P), and mesenchymal cells (M) are indicated. Bar, 50 µm.

View larger version (34K): [in this window] [in a new window]   FIG. 3. Testicular T levels and T production rates after forskolin stimulation in neonatal and adult (+/+) and (-/-) mice. A, Testicular T level in 1- and 70-d-old mice. ***, P < 0.001 compared with WT groups. n = 6–9 mice/group. B, T production after forskolin (5 µmol/liter) stimulation for 4 h. Each bar is the mean ± SEM of four or five individual incubations. **, P < 0.01 compared with (+/+) groups.

Developmental changes in steroidogenesis-related genes and RLF in WT and LuRKO testes
To study further the molecular mechanisms related to the endocrine function of fetal and adult Leydig cells in LuRKO mice, mRNAs encoding several steroidogenesis-related genes as well as RLF were studied during postnatal development. The key gene products in the conversion of cholesterol to T involve the StAR protein, a transporter of cholesterol from the outer to the inner mitochondrial membrane, and specific steroidogenic enzymes, including P450scc, P450-17OH, 17ßHSDIII, and 3ßHSDI. RLF is a specific marker of mature fetal and adult Leydig cells with a known function regulating the trans-abdominal phase of testicular descent (19). Semiquantitative RT-PCR measurements showed that the mRNA levels of StAR, P450scc, P450-17OH, 3ßHSDI, 17ßHSDIII, and RLF were similar in 1-d-old (-/-) and (+/+) testes (Fig. 4). The findings were practically the same on d 5, with the exception of a nonsignificant suppression of P450-17OH. The level of P450scc mRNA was significantly decreased in 10-d-old (-/-) testes, and those of P450-17OH, 17ßHSDIII, StAR, and RLF mRNAs were dramatically reduced from the age of 20 d onward. At the age of 70 d, these mRNAs reached their lowest levels compared with (+/+) testes: P450scc, 3.3%; P450-17OH, 0.03%; 17ßHSDIII, 4.6%; StAR, 15%; and RLF, 7.1%. In striking contrast, the level of 3ßHSDI mRNA was nearly the same in (-/-) and (+/+) testis at all ages studied. No bands were found in control reactions without adding the cDNA, and in RT reaction without AMV-RT in the testicular samples (data not shown), which eliminate RNA or genomic DNA contaminations. Figure 5 shows the age-related expression levels of the same mRNAs in (+/+) and (-/-) testes. The expression levels of P450scc, P450-17OH, 17ßHSDIII, StAR, and RLF were increased in (+/+) testes from d 1 to 5 and were slightly decreased on d 10. From d 20, the levels of all mRNAs were increased again, reaching their maximum level in adulthood. In contrast, in LuRKO testes, the expression levels of the steroidogenic enzyme and RFL genes decreased from the age of 1 or 5 d, and that of StAR decreased from d 20. The different response of 3ßHSDI is apparent, with similar expression levels in both types of testes at all time points.

View larger version (68K): [in this window] [in a new window]   FIG. 4. Semiquantitative RT-PCR detection of mRNAs for several steroidogenesis-related genes during postnatal testicular development in (+/+) and (-/-) mice. A, Representative semiquantitative RT-PCR analyses of P450scc, P450-17OH, 3ßHSDI, 17ßHSDIII, StAR, RLF, and ß-actin mRNAs at different ages in the testes of (+/+) and (-/-) mice. B, Densitometric quantitation of the semiquantitative RT-PCR products of P450scc, P450-17OH, 3ßHSDI, 17ßHSDIII, StAR, and RLF mRNAs. The values are normalized to the intensity of the ß-actin to correct for the differences and are expressed as percentages of the levels measured in (+/+) groups. The results are the mean ± SEM of three independent experiments; the mean of WT controls was assigned a value of 100%. *, P < 0.05; **, P < 0.01; ***, P < 0.001 [compared with (+/+) mice].

View larger version (32K): [in this window] [in a new window]   FIG. 5. Expression of P450scc, P450-17OH, 3ßHSDI, 17ßHSDIII, StAR, and RLF mRNA levels during postnatal testis development in (+/+) and (-/-) mice. Densitometric quantitation of the semiquantitative RT-PCR products of P450scc, P450-17OH, 3ßHSDI, 17ßHSDIII, StAR, and RLF mRNAs was performed. The values were normalized to the intensity of the ß-actin message to correct for the methodological differences and are expressed as percentages of the levels measured in d 1 groups. The results are the mean ± SEM of three independent experiments; the mean of d 1 groups was assigned a value of 100%.

View larger version (36K): [in this window] [in a new window]   FIG. 6. Northern blot analysis of the expression of several steroidogenesis-related genes in the testes of adult (+/+) and (-/-) mice. A, Representative Northern hybridization analyses of P450scc, P450-17OH, 3ßHSDI, 17ßHSDIII, StAR, and RLF mRNAs. 28S ribosomal RNA is shown in the lower panels and used as a loading control. B, Densitometric quantitation of the P450scc, P450-17OH, 3ßHSDI, 17ßHSDIII, StAR, and RLF mRNAs. The values were normalized to the intensity of the 28S RNA to correct for the loading differences and are expressed as percentages of the levels measured in WT groups. The results are the mean ± SEM of three independent experiments; the mean of WT controls was assigned a value of 100%. **, P < 0.01; ***, P < 0.001 [compared with (+/+)].

View larger version (111K): [in this window] [in a new window]   FIG. 7. Representative immunohistochemical locations of P450scc, P450-17OH, and 3ßHSDI during the postnatal development of (+/+) and (-/-) testes. Comparable intensities of P450scc, P450-17OH, and 3ßHSDI immunostainings were observed in the interstitium of (+/+) and (-/-) mice on d 1. Representative fetal Leydig cells (FLC), adult Leydig cells (ALC), peritubular precursor Leydig cells (PL), and mesenchymal cells (M) are indicated. Bar, 50 µm.

View larger version (39K): [in this window] [in a new window]   FIG. 8. Western blot analyses of P450scc, P450-17OH, and 3ßHSDI proteins in (+/+) and (-/-) mouse testes at different ages. Testes from d 1, 20, and 70 WT and (-/-) mice were dissolved in lysis buffer. The protein extracts were separated on 12.5% SDS-PAGE and transferred onto nitrocellulose membrane, followed by detection using anti-P450scc, anti-P450-17OH, and anti-3ßHSDI antibodies.

View larger version (16K): [in this window] [in a new window]   FIG. 9. Expression of TSP2 and 3ßHSDVI mRNA levels in (+/+) and (-/-) mouse testes during the postnatal development. Each bar is the mean ± SEM of measurements of three individual samples.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

During testicular development, two types of Leydig cells, fetal and adult, populate the testis (2, 21). The former appear in fetal life and are responsible for the production of T for masculinization of the male urogenital structures and of RLF for regulating the transabdominal phase of testicular descent (3). These cells regress postnatally, although in the rat some of them may persist until adult life (4, 22). The adult Leydig cells appear at puberty and produce the T required for the onset of spermatogenesis and the maintenance of adult male reproductive functions (23). Testicular steroid hormone biosynthesis is a hormonally regulated, multistep process in which LH is known to play a crucial role as a trophic regulator. Our findings provide convincing evidence that LH/LHR signaling is not essential for the initial differentiation of fetal Leydig cells, as testicular T levels and forskolin-stimulated T production were indistinguishable in the 1-d-old (-/-) and control mice. Moreover, testicular morphology and numbers of Leydig cells were similar in 1-d-old (-/-) and (+/+) mice. However, fetal Leydig cells do express LHR and respond with increased steroidogenesis to LH stimulation (16, 24, 25, 26), but this response seems to be a protective backup mechanism rather than a necessity. Forskolin stimulates adenylate cyclase, which is also the major pathway mediating LH signaling. The rise in T production after forskolin stimulation in fetal Leydig cells in (-/-) mice indicates that the lack of LHR does not affect the downstream signaling system of the receptor.

Leydig cell androgen biosynthesis occurs through concerted action of a cascade of carrier proteins and steroidogenic enzymes. Mutations of any of these components result in deficiency of steroidogenesis (27, 28, 29). Semiquantitative RT-PCR analyses indicated that the levels of all of the measured mRNAs encoding steroidogenesis-related proteins, e.g. P450scc, P450-17OH, 3ßHSDI, 17ßHSDIII, and StAR, as well as RLF were similar in 1-d-old (-/-) and (+/+) mice. Similar amounts of immunopositive cells for P450scc, P450-17OH, and 3ßHSDI were found in control and (-/-) testes at this age, and this was confirmed by Western blot analyses. Taken together, all of the data support the view that steroidogenesis is intact and independent of LH/LHR signaling in mouse fetal Leydig cells. The finding is in agreement with previous studies in which GnRH- and gonadotropin-deficient hpg mice are normally masculinized at birth, and development and function of fetal Leydig cells are normal (11). Likewise, in glycoprotein hormone common -subunit knockout mice, deficient of bioactive LH, FSH, and TSH, sexual differentiation and fetal genital development occur normally (30). The specific reasons for why and how fetal Leydig cells are able to produce high levels of T without LH/LHR signaling remainsunknown, but there is ample evidence that paracrine factors and nongonadotropic hormones play an important role in the early proliferation and differentiation of fetal Leydig cells (12, 24, 25, 26, 31, 32, 33).

In the postnatal life of WT mice, the expression levels of testicular P450scc, P450-17OH, StAR, and RLF genes were high or increased between d 1–5 and slightly decreased by d 10. From d 20 onward, the expression of all of these genes increased again, reaching maximum levels in adulthood, in agreement with a recent study by O’Shaughnessy et al. (20). In contrast, in LuRKO mouse testis, the expression levels of these genes decreased from d 1 onward, attaining very low or undetectable levels in adulthood. The expression levels of P450scc, P450-17OH, 17ßHSDIII, StAR, and RLF on d 5, 10, and 20 were lower in (-/-) testes compared with WT testes of the same age, indicating that fetal Leydig cell differentiation and proliferation are dependent on LHR signaling after birth, as adult-type Leydig cells appear around the second postnatal week (2).

Differentiation of the adult Leydig cell population starts in rodent testes around the second postnatal week (5, 10). This developmental process consists of multiple steps, in which nonsteroidogenic precursor cells in testicular interstitium are transformed into mature Leydig cells with steroidogenic potential through a series of morphological and functional alterations. The role of LH in triggering the initial Leydig cell differentiation is controversial. Several studies have suggested that LH is the triggering hormone for adult Leydig cell differentiation, whereupon this hormone stimulates the differentiation of mesenchymal cells into progenitor cells in the postnatal rat testis (34, 35). During the testicular development of LuRKO mice, positive immunostaining for P450scc and 3ßHSDI was found in mesenchymal cells and peritubular Leydig precursor cells from d 10 onward, and low levels of mRNA expression for these genes were detected by RT-PCR. This observation is important because it indicates that mesenchymal cells and peritubular Leydig precursor cells attain low levels of steroidogenic activity, and the onset of adult Leydig cell differentiation is independent of LH action. Previous studies reported that when adult Leydig cells first appear in the neonatal rat testis, circulating LH is very low (36), and after transient neonatal hypothyroidism, Leydig cells still differentiate in the presence of very low LH levels (37, 38). It has also been reported that during Leydig cell regeneration in ethane-dimethane-sulfonate (EDS)-treated adult rats, precursor cell proliferation and transformation into progenitor cells occur without LH, as demonstrated by hypophysectomy or using T implants before EDS treatment (39). Ariyaratne et al. (40) reported that mesenchymal cells become steroidogenically active before attaining LHR expression. Hence, the onset of mesenchymal cell differentiation into Leydig cells in the prepubertal rat is not dependent on LH. Our present findings taken together with previous studies suggest that LH is not crucial in this initiation process. However, the factor(s) triggering mesenchymal cell differentiation into Leydig cells in the postnatal mouse testis remain unknown. A recent study showed that hypothyroidism inhibits Leydig cell regeneration, and hyperthyroidism results in accelerated differentiation of mesenchymal cells into Leydig cells after their depletion by EDS treatment, indicating that thyroid hormone may be an important initiator of Leydig cell differentiation (13).

A recent study demonstrated that TSP2 is mainly expressed in fetal Leydig cells, and 3ßHSDVI and 17ßHSDIII are expressed in adult Leydig cells, thus being markers of two different types of Leydig cells (14, 20, 41). Our real-time and semiquantitative RT-PCR data showed that TSP2 is predominantly present in (-/-) adult testes along with very weak or undetectable 3ßHSDVI and 17ßHSDIII expression, indicating that the Leydig cells present in (-/-) testes have at least some features of the fetal population. This may indicate that the disappearance of fetal Leydig cells during postnatal development may be slower in (-/-) than in (+/+) testes.

Interestingly, the expression of 3ßHSDI mRNA and protein was detected at all ages studied, and it was at the same level in WT and (-/-) mice, indicating that the expression of this gene is independent of LH/LHR signaling, in agreement with a previous study (14). The reason for the constant levels of 3ßHSDI mRNA and protein is that this gene is expressed in mesenchymal and peritubular Leydig precursor cells in the absence of mature Leydig cells in adult (-/-) testis. Previous studies have shown that 3ßHSDI was detected as early as d 13 postcoitum throughout fetal development, even before sexual differentiation (42, 43). The role of 3ßHSDI at the early stages of development and in the precursor Leydig cells of LuRKO mice is still not clear. All of the steroidogenesis-related genes, except for 3ßHSDI, were similarly affected by the absence of LH action in adulthood. P450-17OH, 17ßHSDIII, and RLF were the most sensitive, and their levels were undetectable in adult testis by Northern blot. This difference may be due to the Leydig cell precursors expressing variable amounts of steroidogenic enzymes.

In conclusion, the LuRKO mouse is an excellent model for the study of Leydig cell development and steroidogenesis. Our findings provide the first specific evidence that the initial differentiation of fetal Leydig cells is not dependent on LH/LHR signaling, but LHR signaling is important for Leydig cell differentiation after birth. However, the initiation of adult Leydig cell differentiation in the mouse testis can begin independently of LH action.

	Acknowledgments

 
We thank J. Vesa and T. Laiho for their skilful technical assistance.

	Footnotes

 
This work was supported by grants from the Academy of Finland, the Sigrid Jusélius Foundation, and the Wellcome Trust.

Abbreviations: AMV-RT, Avian myeloblastosis virus reverse transcriptase; EDS, ethane-dimethane-sulfonate; FSHR, FSH receptor; 3ßHSDI, 3ß-hydroxysteroid dehydrogenase I; 17ßHSDIII, 17ß-hydroxysteroid dehydrogenase type III; LHR, LH receptor; LuRKO, LH receptor knockout; P450-17OH, 17-hydroxylase cytochrome P450; P450scc, cytochrome P450 side-chain cleavage; RLF, relaxin-like factor; SDS, sodium dodecyl sulfate; SSC, standard saline citrate; StAR, steroidogenic acute regulatory protein; T, testosterone; TBS, Tris-buffered saline; TSP2, thrombospondin; WT, wild-type.

Received August 13, 2003.

Accepted for publication November 19, 2003.

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