Hum. Reprod. Advance Access originally published online on December 16, 2005
Human Reproduction 2006 21(4):936-942; doi:10.1093/humrep/dei433
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Cryptorchidism in LhrKO animals and the effect of testosterone-replacement therapy
Division of Research, Department of Obstetrics, Gynecology & Women’s Health, University of Louisville, Health Sciences Center, Louisville, KY, USA
1 Present address: Fujian Medical University, 88 Jiao Tong Road, Fuzhou, Fujian 35004, China
2 To whom correspondence should be addressed at: Department of Obstetrics, Gynecology & Women’s Health, 438 MDR Building, University of Louisville, Health Sciences Center, Louisville, KY 40292, USA. E-mail: zhenmin.lei{at}louisville.edu, cvrao001{at}louisville.edu
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Abstract |
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BACKGROUND: The objective of the current study was to characterize the morphological and genetic basis of cryptorchidism. METHODS AND RESULTS: We investigated cryptorchidism in LH receptor (Lhr) knockout (LhrKO) mice and how testosterone-replacement therapy (TRT) worked to correct the phenotype. The results revealed that while gubernacular development was indistinguishable between Lhr-null and wild-type animals until 7 days of age, it was subsequently severely impaired in null animals. This was due to a reduction in mesenchymal cell division, differentiation into cremaster muscle cells and their delayed maturation. While transcript levels of Hoxa10, Hoxa11, Desrt and Dll1 were indistinguishable, the levels of Notch1, Numb and Lgr8 in the gubernaculum and Insl3 in the testes were lower in Lhr-null than in wild-type siblings. The TRT, which completed testicular descent into the scrotum, corrected the morphological changes and the expression of Lgr8, Numb and Notch, but not Insl3, to wild-type levels. Transection of the genitofemoral nerve did not prevent the TRT effect. CONCLUSION: In summary, cryptorchidism in Lhr-null animals was caused by defects in the gubernacular development due to testosterone deficiency. TRT reversed all the morphological and gene expression changes except Insl3, suggesting that testosterone, not INSL3, secreted by Leydig cells, facilitates the completion of testicular descent.
Key words: cryptorchidism/gene expression/gubernaculum/LhrKO/testosterone therapy
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Introduction |
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Testicular descent into the scrotum takes place in two phases. In the first, transabdominal phase, testes move into the internal inguinal ring during embryogenesis between 15.5 and 17.5 days of pregnancy in mice (Zimmermann et al., 1999
). Genetic evidence suggests that this phase is dependent on both androgens and insulin-like factor 3 (INSL3, also known as relaxin-like factor), secreted by fetal-type Leydig cells. While androgens cause regression of the cranial suspensory ligament (CSL), INSL3, binding to leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8, also known as relaxin receptor 2) is responsible for outgrowth of the gubernaculum which involves cell proliferation and differentiation (Nef and Parada, 1999; Zimmermann et al., 1999; Tomiyama et al., 2003; Feng et al., 2004). In the second phase, testes migrate into the inguinal region and then into the scrotum during the third postnatal week in mouse. During this migration, mesenchymal cells in the gubernacular bulb gradually disappear, while the newly formed muscle layer invaginates and grows caudally into the space of the mesenchymal core. This invagination in the direction of the developed scrotum forms the processus vaginalis, which is also called the cremaster sac (Hutson et al., 1997). The mechanism that regulates the second phase of testicular descent is not completely understood (Ivell and Hartung, 2003). For example, it is debated whether regression of the gubernaculum allows the formation of the cremaster sac or whether the gubernaculum actively proliferates and differentiates into a new cremaster muscle (Wensing, 1988; van der Schoot, 1996; Hrabovszky et al., 2002). Nevertheless, this phase is androgen dependent. However, the site of androgens action, i.e. gubernaculum and/or genitofemoral nerve (GFN), is not clear (Husmann and Levy, 1995).
Targeted disruption of the LH receptor gene (Lhr) had no effect on prenatal sexual differentiation and gonadal development (Lei et al., 2001). However, it impaired the postnatal testicular growth and development. The Lhr-null testes contain mostly fetal-type and few, if any, adult-type Leydig cells. As a result, serum testosterone levels were greatly reduced. Adult null males have bilateral cryptorchid testes, located slightly above the bladder neck. There was no evidence of persistent CSL, indicating that prenatal androgens may have already caused its regression. The testis and epididymis were attached by the gubernacular cord to the inguinal region, but in no case did testes descend into the scrotum. Twenty-one-day testosterone-replacement therapy (TRT) of 30-day-old null animals resulted in testicular descent into the scrotum (Lei et al., 2001, 2004; Rao and Lei, 2002). In the present study, we systematically investigated the postnatal morphological development of the gubernaculum and gene expression changes that account for cryptorchidism in Lhr-null animals and the effect of TRT on morphological and gene expression changes.
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Materials and methods |
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LhrKO mice
Generation of the Lhr knockout (LhrKO) mice has previously been described (Lei et al., 2001
). Mice were maintained as required under the National Institutes of Health guidelines for the Care and Use of Laboratory Animals. All studies have been approved by the Animal Care and Use Committee of the University of Louisville. Adult male and female heterozygous mice were mated to obtain wild-type (+/+), heterozygous (+/–) and homozygous (–/–) animals. The genotype was determined by PCR with tail genomic DNA and Lhr and neomycin-resistance gene (neo) primers [Lhr: 5'-TGACCTGTTCCTGGGGCT GCTG-3' (forward), 5'-AAATGGCCTCAACGGGTGTGCA-3' (reverse); neo: 5'-CGGAAGCCCGGCATTCTGCA-3' (forward), 5'-ATTCAG GGCACCGGACAGGTCG-3' (reverse)]. The Lhr primers were designed to detect a 284 bp DNA fragment of the 5'-flanking region of a wild-type Lhr gene. This region was deleted and replaced by a neo cassette in the mutant gene. Thus, PCR performed on each sample with these two sets of primers allowed unambiguous identification of all three genotypes. All animals were maintained on 12-h light–dark cycles with food and water provided ad libitum. Three to six 1-, 7-, 14-, 30- and 60-day-old mice of each genotype (day of birth was designed as day 1) were used for each experiment.
TRT
Twenty-one-day time-release pellets containing 5 mg testosterone (Innovative Research America, Sarasota, FL, USA) were subcutaneously implanted into null animals at 30 days of age, which corresponds to puberty when circulating testosterone levels normally begin to rise. The pellets represent a ‘Matrix Driven Delivery’ system integrating the principles of diffusion, erosion and concentration gradient resulting in a biodegradable matrix that effectively and continuously releases testosterone. The therapy resulted in an increase in serum testosterone levels to about 800±100 ng/dl (Lei et al., 2001; Rao and Lei, 2002; Lei et al., 2004). Null mice for controls were implanted only with placebo pellets. The age of animals at the end of therapy corresponded approximately to the age of sexual maturity (6–8 weeks), and they were killed to recover the testes and gubernacula.
Histology and computerized quantitative morphometry
The animals were deeply anaesthetized by intraperitoneal injection of sodium pentobarbital. For 1-, 7- and 14-day-old mice, the lower body was hemicorporectomized, fixed in 10% buffered formalin and embedded in paraffin. Serial sagittal 5 µm sections were cut for haematoxylin and eosin (H&E) and immunohistochemical staining. For 30- and 60-day-old mice, the abdominal cavity was opened by a sagittal incision. The testes and gubernacula were dissected. The tissues were either fixed in 10% buffered formalin for H&E and immunohistochemical staining or stored at –80°C for RNA isolation. H&E-stained serial sections were examined under bright-field microscope for quantitative morphometry using a computerized Bioquant IV System (R & M Biometrics INC, Nashville, TN, USA) and photography. Computer images of the sagittal vertical sections of gubernaculum with the caudal of gubernaculum attaching to the tip of the cremaster sac were used for all the measurements. The tip width of the cremaster sac was defined as the thickness of the tip where the gubernaculum remains in contact with the scrotal floor (Figure 1B). The end width of the cremaster sac was defined as the thickness of the cremaster muscle that ends at the abdominal rectus muscle (Figure 1B). The areas of the cremaster muscle (Figure 1B) and gubernaculum cord (Figure 1B) were also measured in the sagittal vertical section of the cremaster sac.
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Immunohistochemistry
This procedure was performed by an avidin–biotin immunoperoxidase method (Vector Laboratories, Burlingame, CA, USA) as described previously (Lei et al., 2001). Briefly, deparaffinized gubernacular sections were treated for 10 min with 3% H2O2 in methanol to block endogenous peroxidase activity. All sections were incubated with 1 : 100 diluted primary antibodies overnight at 4°C. Polyclonal antibody to proliferative cell nuclear antigen (PCNA) and monoclonal antibodies to embryonic, neonatal and adult myosin heavy chain were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the Department of Biological Sciences, the University of Iowa (Iowa City, IA, USA), respectively. Substitution of primary antibodies with nonspecific immunoglobulin G served as procedural controls.
Gubernacular apoptosis assay
TdT-mediated dUTP nick-end label staining was performed on formalin-fixed and paraffin-embedded sections using a DeadEnd colorimetric apoptosis detection system (Promega, Madison, WI, USA) as described previously (Lei et al., 2004). The –/– testes that contained many apoptotic cells were included as a positive procedure control. The brown coloured nuclei of apoptotic cells in all parts of the gubernacula were examined under a bright-field microscope.
GFN transection
The surgery was performed essentially according to the procedures described by Beasley and Hutson (1987) and Fallat et al. (1992). Briefly, 20-day-old +/+ and –/– mice were anaesthetized by intraperitoneal injection of sodium pentobarbital, and then a small longitudinal incision was made through the left lateral abdomen wall above the inguinal canal. The left ureter was identified and used as an anatomical marker to track the GFN. The GFN was transected as it crossed the anterior part of the psoas muscle with ophthalmic scissors. The transection level was at L3, which is above the point of division into femoral and genital branches. The right GFN was left intact as an internal control in each animal. The incisions were closed by stitches. Seven days after surgery, animals were placed on placebo or TRT for 21 days. The animals were killed at 48 days of age for collection of both left and right gubernacula. The scrotal or abdominal location of the testis at the time of removal was noted.
Semiquantitative RT-PCR
The RNA isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) was reverse transcribed using avian myeloblastosis virus (AMV) reverse transcriptase (Promega) and random oligonucleotide hexamers (Invitrogen). PCR primers, as listed in Table I, were designed according to the sequences obtained from Genebank using the Vector NTI 9.0 computer program (InforMax, Frederick, MD, USA) and synthesized by Operon Technologies (Alameda, CA, USA). All the primers were designed to amplify products that covered one or more exons.
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Twenty-five microlitres of total reaction mixture [3 µg of total RNA, 1.5 µg of random primer, 40 units Rnasin® plus RNase inhibitor (Promega) and 30 units AMV] was incubated for 60 min at 37°C and then for 5 min at 95°C. Then PCRs were performed with primer sets for target genes and a housekeeping gene primer set (Table I) of ribosomal protein large subunit 19 (Rpl19) as an internal control for both complementary DNA (cDNA) quantity and quality. The reaction mixture contained 1.0 µM of forward and reverse primers, 2 mM MgCl2, 1 µl cDNA, 200 µM dNTPs and 1.5 units of Taq DNA polymerase (Promega). Thirty-four cycles for Delta-like 1 (Dll1), 28 for homeobox protein A 10 (Hoxa10), developmentally and sexually retarded with transient immune abnormalities protein (Desrt) and numb homologue (Numb, an adaptor of Notch1), 30 for Hoxall and Lgr8, 31 for notch homologue 1 (Notch1, the receptor for Dll1) and 26 for Insl3, which were in the linear phase of PCR amplification, were used. Each cycle consisted of denaturation for 45 s at 94°C; annealing for 45 s at 57°C; extension for 75 s at 72°C and the last extension for 5 min at 72°C. The PCR amplified products were electrophoresed in 1.5% agarose gels, stained with ethidium bromide and analysed using the TotalLab V 2.01 (Nonlinear USA, Durham, NC, USA) image analysis software. Then, the ratio of target gene to housekeeping gene was calculated. All the reactions were repeated two to three times on three samples in each genotype. Procedural controls included omissions of RNA or cDNA templates, reverse transcriptase or random primers.
Statistical analysis
The data presented are the means ± SEM. All results were analysed by one-way analysis of variance and Tukey multiple comparison post test using an Instat Version 3.06 program (Graphpad Sofware, San Diego, CA, USA). A P-value <0.05 was considered significant.
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Results |
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Morphological analyses of postnatal development of the gubernaculum
Figure 1A shows that gubernacular cord was small and flat and cremaster sac was too small to accommodate testis in 60 day-old –/– animals as compared with +/+ and +/– siblings (Figure 1A, arrow). Twenty-one-day TRT completely normalized the gubernacular growth and development and the size of the cremaster sac in –/– animals (Figure 1A).
Histological examination of serial sagittal sections revealed that there were no morphological differences between +/+ and –/– animals in the gubernacular development until 7 days after birth (Figure 1B–E). In both genotypes, the gubernacular bulb of 1-day-old animals primarily contained mesenchymal cells (Figure 1B,C). Subsequently, the core bulb area became clear and mesenchymal cells began to differentiate into muscle cells. Their cytoplasm was abundant and eosinophilic and was similar to that in the cremaster sac. Beginning 14 days after birth, most mesenchymal cells in the bulb differentiated into muscle cells in +/+ animals based on the morphology and staining property. At the leading edge, a clear space was often seen between gubernaculum and the scrotal floor (Figure 1F,H, open arrow). The bulb size gradually diminished from 30 days of age and completely disappeared in 60-day-old +/+ animals (Figure 1H,J). The gubernacular developmental defect became obvious from 14 days of age in –/– animals (Figure 1G,I,K). The gubernacular bulb of these animals contained undifferentiated basophilic mesenchymal cells (Figure 1I, open arrow) which persisted into adulthood.
Quantitative morphometric analysis demonstrated that +/+ and –/– animals had a similar tip and end width of the cremaster sac until 7 days of age (Figure 2A,B). Subsequently, –/– animals had a lower end width from 14 days of age, and the difference compared with +/+ siblings became even more dramatic with age. The lower tip width was first seen in 30-day-old –/– animals, and it persisted as the animals aged. The cremaster muscle area was lower at 14 days of age and it continued through 60 days of age in –/– animals (Figure 2C). The gubernacular cord area was lower from 30 days of age in –/– animals (Figure 2D). Twenty one-day TRT normalized all the morphological defects (Figure 1L, Figure 2A–D).
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PCNA immunostaining during postnatal development of the gubernaculum
The immunostaining for PCNA, a cell proliferation marker, was present in the nuclei of interstitial and cremaster cells of the gubernaculum (Figure 3). The positive stained cells were abundant from postnatal days 1–14, and then they decreased in +/+ animals (Figure 3A–I). The PCNA immunostaining pattern was indistinguishable between –/– and +/+ animals until 7 days of age. Subsequently, null animals showed a marked decrease by day 14. The genotype differences beyond day 14 were either subtle or nonexistent (Figure 3G,H,J,K). Regardless of the age and genotype, gubernacula contained very few apoptotic cells (data not shown).
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Effect of GFN transection on the gubernacular development and inguinoscrotal testicular descent
To determine whether GFN was involved in TRT-induced testis descent in null animals, it was transected on the left side, and the right side was kept intact in 20-day-old animals. In –/– animals placed on TRT and in +/+ siblings, the testis descended into the scrotum in the GFN transected side, just as in the intact side. Appearance of testis and gubernacula on both sides was also similar. GFN transection, however, resulted in a smaller psoas muscle on the left side than on the right side, indicating that the GFN transection was complete, and it was above the point of division into femoral and genital branches.
Developmental changes in embryonic, neonatal and adult myosin immunostaining in cremaster muscle
Immunostaining for embryonic and neonatal myosins decreased and for adult myosin increased from 30 to 60 days of age in +/+ animals (Figure 4). Both embryonic and neonatal myosin immunostaining also decreased in –/– animals, but was not as dramatic as in +/+ siblings. As a result, 60-day-old –/– animals contained higher levels than +/+ siblings (Figure 4). Although the adult myosin immunostaining intensity was not different between –/– and +/+ animals, the cremaster muscle was much thinner in –/– animals than in the +/+ siblings.
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Messenger RNA levels of genes associated with testicular descent and cremaster muscle development
The gubernacular messenger RNA levels of Hoxa10, Hoxa11, Desrt and Dll1 were similar between –/– and +/+ animals (Figure 5). Semiquantitative RT-PCR analysis showed a significantly lower Notch1, Numb, Lgr8 and Insl3 mRNA levels in –/– animals than in +/+ siblings. TRT, however, restored these levels to wild type, except Insl3 (Figures 5 and 6).
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Discussion |
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Cryptorchidism is a birth defect with consequences of torsion, testicular cancer and infertility. Unless it is spontaneously resolved during the first few months of life, surgical correction is required (Ivell and Hartung, 2003
; Adham and Agoulnik, 2004). A better understanding of this birth defect could come from animal models, and several of them already exist. All the models have either decreased testosterone levels, or interference with testosterone action, or mutation or inactivation of downstream target genes (Hutson et al., 1997; Ivell and Hartung, 2003; Klonisch et al., 2004). LhrKO animals are a new addition to this model list, which offers validation and further exploration to test how testosterone works in inducing testicular descent during the transinguinal phase.
Morphological analysis of gubernacular development revealed that it was normal until 7 days after birth in –/– animals. Subsequently, it lagged behind the +/+ siblings. As a result, the size of the cremaster sac in adult null animals was about the same as in 1-week old wild-type animals. The lack of development is due to a decrease in mesenchymal cell proliferation, their differentiation into muscle cells and delayed maturation. Cellular apoptosis does not seem play a significant role in these events. A 21-day TRT regimen on 30-day-old –/– animals normalized gubernacular development due to an increase in cell proliferation, differentiation into muscle cells and cremaster muscle maturation. Thus, these results reaffirm that LH-driven Leydig cell testosterone production is necessary for second phase of testicular descent (Ivell and Hartung, 2003; Klonisch et al., 2004). Transinguinal migration of testis into the scrotum normally occurs between the second and third postnatal weeks in mice (Hutson et al., 1997; Zimmermann et al., 1999). The fact that TRT administration was able to induce apparently normal testicular descent in 30-day-old animals suggests that the window of the final stage of testicular descent is not just restricted to the second and third postnatal weeks. However, whether it still remains open into late adulthood is not known.
We investigated the potential testosterone action on GFN, as previous studies have shown that its transection in newborn rats caused a defective second phase of testicular descent (Shono et al., 1999). If intact GFN is necessary for testosterone effect, then its transection should prevent testosterone action in Lhr-null animals. When unilateral transections were performed, testicular descent proceeded normally on the transected side just as in the intact side in null animals placed on TRT and in +/+ siblings. Therefore, the discrepancy between the present findings and previous reports could be related to the transection timing and/or reflection of a unique species difference.
Testosterone works by activating its receptors, which are present in cremaster muscle and other structures associated with testicular descent (Husmann and McPhaul, 1991; Yuan et al., 2004). The receptor activation results in the upregulation of several target genes. Several genes have previously been inactivated to determine their importance in testicular descent. The expression of genes such as Hoxa10, Hoxa11 and Desrt, which are thought to be important for testicular descent (Hsieh-Li et al., 1995; Satokata et al., 1995; Lahoud et al. 2001), was neither decreased nor increased by TRT in gubernaculum of –/– animals. Therefore, these genes must be considered independent of testosterone action. INSL3, which is secreted by Leydig cells, and its receptor in gubernaculum, LGR8 (Kumagai et al., 2002; Adham and Agoulnik, 2004), dramatically decreased. TRT, which induced testicular descent, did not restore Insl3 expression, which was due to the absence of adult-type Leydig cells in the testis of adult –/– animals. Its receptor, Lgr8 in gubernaculum, was however restored by TRT. These findings suggest that primary testosterone action involves maintaining normal LGR8 levels so that even very low INSL3 levels present in null animals can activate them. Alternatively, there could be other peptides like relaxin which can also activate LGR8 (Hsu et al., 2002). Congenital mutation or targeted disruption of either Insl3 or Lgr8 genes demonstrated that they were essential for the first phase of testicular descent (Ferlin et al., 2003; Adham and Agoulnik, 2004; Foresta and Ferlin, 2004). The present study suggests that LGR8 signalling may also play an important role in the second phase of testicular descent.
Activation of DLL1 receptor by NOTCH1 results in an increase in proliferation of muscle precursor cells. The NUMB, a NOTCH1 adaptor, is known to regulate cell differentiation and cell fate determination during postnatal myogenesis (Shawber et al., 1996; Nofziger et al., 1999; Delfini et al., 2000; Hirsinger et al., 2001). Among these three genes, only Notch1 and Numb mRNA levels were lower in –/– animals than in +/+ siblings and TRT restored their levels in null animals. Thus, Notch1 and Numb, rather than Dll1, may play an important role in testicular descent induced by androgens.
In summary, defective cell proliferation, differentiation and myogenesis in the gubernaculum and decreased expression of some but not all of the genes associated with testicular descent were responsible for cryptorchidism in Lhr-null animals. Testosterone, not INSL3, secreted by adult-type Leydig cells, through its GFN-independent actions, facilitates the completion of the second phase of testicular descent by maintaining normal cell proliferation and differentiation via Numb, Notch1 and Lgr8 gene expression.
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Acknowledgement |
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We gratefully acknowledge the technical ssistance of Qingbo Lu and Fred Carman Jr. This work was supported by NIH grant, HD-40223.
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References |
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Submitted on October 7, 2005; resubmitted on November 11, 2005; accepted on November 21, 2005.
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