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Regular Article |
a Southern Illinois University School of Medicine, Department of Physiology, Carbondale, Illinois 62901
b Curis, Inc., Cambridge, Massachusetts 02138
ABSTRACT
Desert hedgehog (Dhh) is a cell-signaling molecule that was first discovered in Drosophila. A unique testicular phenotype has been described in neonatal and adult Dhh-null animals that includes anastomotic seminiferous tubules, pertitubular cell abnormalities, and absence of adult-type Leydig cells. In the present study, we addressed the developmental basis for the abnormalities previously described for the adult Dhh-null phenotype. The source of Dhh is the Sertoli cell, and receptors are localized on peritubular cells and possibly Leydig cells. The development of testes from Dhh-null mouse embryos was studied using light and electron microscopy at 11.5, 12.5, 13.5, and 16.5 days postcoitum (dpc) and was compared with that in control Dhh heterozygous and wild-type embryos. Dhh-null and control testes were generally similar during the period of early cord formation (11.5–12.5 dpc). By 13.5 dpc, the basal lamina delimiting the cords was lacking in some regions and disorganized in Dhh-null testes, and occasional germ cells were seen outside cords. At 16.5 dpc, these defects were more prominent and cord organization was less well defined than in controls. In addition, there were numerous extracordal germ cells, some of which were partially enclosed by a somatic cell of unknown identity. Numerous fibroblast-like cells, apparently secreting collagen and basal lamina, characterized the interstitium of the Dhh-null testis. These defects likely stem from abnormal peritubular stimulation due to the lack of Dhh, leading to the abnormalities seen in the developmental stages studied here and in the adult testis.
developmental biology, embryo, interstitial cells, Sertoli cells, spermatogenesis
INTRODUCTION
Proper cell-cell signaling is required for normal patterning and development during embryogenesis. A number of signaling factors have been characterized relatively recently, including the Drosophila gene hedgehog (hh), which is important for segment polarity [1–4]. In mammals, three hedgehog genes and their products have been described: Desert hedgehog, Indian hedgehog, and Sonic hedgehog [5].
Expression of Dhh has been studied by in situ hybridization and was reported to be expressed as early as 11.5 days postcoitum (dpc) in Sertoli cells of the undifferentiated male gonad and in Schwann cells along peripheral nerves. Minor expression was associated with the developing atrioventricular valve of the heart and endothelial cells of blood vessels. No expression was observed in the ovary [6, 7]. Because the transcription factor Sry is expressed by 10.5 dpc in the testis and Müllerian inhibiting substance (MIS) is the only other male-specific gene known to be expressed as early as 11.5 dpc, Dhh could be a gene that is modulated by Sry, downstream of its expression. In addition, Dhh expression is present in Sertoli cells during later developmental stages and persists in the adult seminiferous tubules. Dhh-null female mice are fertile, but males have relatively small embryonic gonads by 18.5 dpc and are sterile into adulthood [7].
The importance of Dhh in testicular morphology was described previously [8] in a report of two testicular phenotypes in adult and neonatal Dhh-null male mice. The majority of the Dhh-null males were pseudohermaphrodites, i.e., feminized males with a blind vaginal opening and evidence of teats. These mice showed undescended and extremely small testes embedded in the fat pads located near the kidneys. Testes contained seminiferous tubules that were irregular and anastomotic because of a major defect in the peritubular tissue. The parietal lymphatic cells lining the seminiferous tubules were frequently absent, the myoid cells were immature in appearance, and the basal lamina facing the Sertoli cells was discontinuous. The interstitial space contained fetal Leydig cells and fibroblast-like cells, with abundant intervening collagen. In testes from newborn animals, gonocytes were largely present within the seminiferous cords, but many gonocytes were also seen outside the cords. The remainder of the Dhh-null males, termed masculinized, were also infertile and showed abnormal peritubular tissue. The parietal endothelial cells of the lymphatic space were often discontinuous. [8]. Spermatogenesis was severely depressed in both phenotypes, with testes containing rare spermatogonia and spermatocytes.
The receptor for the hh proteins is Patched (Ptc) [9–13]. In the mouse embryo, ptc was reported to be expressed in the male but not the female gonad at 11.5 dpc, which was after the onset of Dhh expression at 10.5 dpc. After formation of the seminiferous cords, expression of ptc was limited to Leydig cells [7]. More recently, however, we localized expression of ptc in the adult and juvenile mouse testis to the peritubular cells, which were the cells most affected by the absence of Dhh in the Dhh-null mice [8]. In the present study, we examined the embryonic testis of the ptc-LacZ mouse, a "knock-in" mouse that has nucleus-targeted expression of LacZ in cells that express ptc [14], to pinpoint the interstitial expression of ptc that has been reported previously [7].
Our previous examination of the Dhh-null phenotype revealed that the lack of Dhh action was already manifested at birth and that the defects had a developmental basis. Given the embryonic expression of Dhh, it appeared to be a gene that is active early in testicular development. In this study, we addressed the developmental aspects of the Dhh-null phenotype to determine when the defects appear so the primary cause of the postnatal defects could be identified.
MATERIALS AND METHODS
Embryos
Dhh-null mice (original breeding stock kindly provided by Dr. Andrew McMahon, Harvard University, Cambridge, MA) were generated as described by Bitgood et al. [7]. For the present study, Dhh homozygous-null or heterozygous females were bred to Dhh heterozygous males. All mice were from a mixed genetic background (129/Sv, C57BL/6, and Swiss Webster). Pregnant females were killed, and embryos were obtained at 11.5, 12.5, 13.5, and 16.5 dpc. Embryos were dissected in PBS, and the gonads and tails were collected. Tail samples were processed for genotyping via polymerase chain reaction for Dhh and also for Sry as a male-specific marker [8]. Control embryos were either Dhh heterozygotes or wild type.
Heterozygous ptc-LacZ males [14] (original breeding stock kindly provided by Dr. Matthew Scott, Stanford University, Palo Alto, CA) were mated with B6D2F1/J females (Jackson Laboratory, Bar Harbor, ME) to generate timed pregnancies. Females were killed at 14.5 dpc, and embryos were collected and dissected in PBS.
All experimental protocols involving animals were approved by the Institutional Animal Care and Use Committee and were in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Histologic Preparations
Gonads from control and Dhh-null embryos were immersion fixed in a 0.1 M Na-cacodylate buffer containing 1 mM CaCl2 and 4% glutaraldehyde. They were subsequently postfixed in an osmium:ferrocyanide mixture, dehydrated in ethanol, infiltrated with propylene oxide, and embedded in epoxy resin [15]. For electron microscopy, thin sections showing silver-gold interference colors were prepared. Sectioning was always made transverse to the long axis of the embryonic gonad. Gonads from ptc-LacZ embryos were fixed and stained as described previously [8] and embedded in Araldite 502 (Electron Microscopi Sciences, Fort Washington, PA). Sections (5 µm) were mounted on glass slides, and photomicrographs were taken through oil at 100x magnification.
Transmission Electron Microscopy
A minimum of two Dhh-null and two control embryos at each of the four developmental ages were analyzed. Thin sections were mounted on coated grids, stained, and photographed at the minimum magnification obtainable by the Hitachi H500 electron microscope (Hitachi, San Jose, CA). Large montages were constructed to allow ultrastructural visualization of the cross-sectioned gonads. Over 1000 electron micrographs were obtained for analyses.
The basal lamina was suspected to be abnormal in Dhh-null embryos and was outlined heavily on the photomicrographs with an erasable grease pencil. Montages were magnified 2.6x the negative magnification but were too large to be published in the space allotted by a single journal page. Therefore, a photocopy machine was used to reduce marked montages to a size where the outlined basal lamina and germ cells could be traced to tracing paper. The tracing paper was scanned for preparation of a computer-generated image, and a scaled drawing was made using a computer graphics program (Adobe Illustrator, Adobe Systems, Salinas, CA).
RESULTS
11.5 dpc
At 11.5 dpc, no differences were noted in the structure of male gonads of control versus Dhh-null embryos (data not shown). A distinct basal lamina was not found in 11.5-dpc male gonads, but some intercellular spaces were darkened slightly, and these appeared to constitute a presumptive basal lamina (not shown).
12.5 dpc
At 12.5 dpc, only minor differences were noted on comparisons of montages (Fig. 1). In the control male gonads, the basal lamina consisted of a thin (7–12 nm across) amorphous layer that could be recognized by electron microscopy (Fig. 2). In some regions, an apparent separation of the presumptive interstitium and seminiferous cords was noted based on the alignment of cells adjacent to and continuing away from the existing basal lamina and a slight densification of the intercellular space in this region (Fig. 3). This logical separation of cells and slight intercellular densification may have been equivalent to the region of laminin deposition that delineated the presumptive cords at 12.5 dpc [16]. Hereinafter, we refer to the logical expected continuation of the basal lamina as the presumptive separation of cordal and interstitial tissue. In both the control and Dhh-null animals, the presumptive cords could only be traced for relatively short distances because there were many regions where the future cordal and interstitial regions could not be readily deduced.
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Also at 12.5 dpc, germ cells were generally associated with or relatively close to one aspect of the basal lamina, i.e., that portion away from the gonadal surface. For the most part, germ cells were situated in or near the concavity of a profile of the basal lamina (Fig. 1). The degree to which the basal lamina was fragmented was always greater in Dhh-null embryos than in control embryos (Fig. 1), although this was not a prominent diagnostic feature.
13.5 dpc
In 13.5-dpc control embryos, electron microscopic evaluation revealed that the basal lamina was nearly continuous to form cords (Fig. 1C). The amorphous material in the basal lamina was slightly thicker (10–15 nm) than that of 12.5-dpc embryos, making the basal lamina more prominent than that of the younger embryos (Fig. 4). Some flattened cells that would presumably develop into myoid cells were found outside the cords (Fig. 4). Similar to the 12.5-dpc embryos, the positioning of cells was such that they extended laterally from the basal lamina, forming an early separation between the presumptive cords and the presumptive interstitium.
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In the 13.5-dpc Dhh-null embryos, some basal laminae ended abruptly without the suggestion of a separation between the presumptive cords and presumptive interstitium (Fig. 5). Short, irregular, fragmented segments of basal laminae and associated collagen were commonly seen and were not attached to the longer stretches of basal lamina forming the cords (Fig. 5). Occasional germ cells were located outside of seminiferous cords or presumptive cords (Fig. 1). Some germ cells within cords were seen at the edge of or bulging from the presumptive cords, giving the impression that they were neither fully within nor fully outside of the developing cords (Fig. 6).
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A closer examination of the basal lamina of 13.5-dpc male gonads revealed that although the basal lamina was relatively uniform in control embryos, the basal lamina of Dhh-null embryonic testes was regionally thicker or thinner than normal (Fig. 7). Numerous areas contained small gaps in the continuity of the basal lamina (Fig. 7) (not shown on the composite drawings, Fig. 1D). Some of these gaps were occupied by pseudopodial processes of germ cells, giving the impression that these germ cells were migrating to the outside of the cords (Fig. 8). Several germ cells were seen extracordally (Fig. 1D).
16.5 dpc
At 16.5 dpc, a prominent and generally uniform basal lamina was continuous around seminiferous cords in control embryos. An inner amorphous layer (approximately 15–25 nm thick) and an outer collagenous layer could be identified (Fig. 9). Interstitial and cordal spaces were well defined, and there were no foci of basal lamina or collagen in the interstitium and no germ cells positioned outside the cords.
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In 16.5-dpc Dhh-null male gonads, there were numerous small discontinuities in the basal lamina (Fig. 10) (not shown on the drawing of the montage, Fig. 1F). Regions could be found where the basal lamina was not complete (Fig. 10) or was relatively thin or thick (Figs. 11 and 12). Basal laminae of the cords often split or took irregular configurations (Fig. 12). Also, there were numerous foci of basal lamina or collagen that were located outside of the cords, positioned around undifferentiated cells that were similar in appearance to fibroblasts (Figs. 11 and 13).
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ptc-LacZ "Knock-In" Embryonic Testes
Nuclear expression of LacZ (blue staining) was used to identify cells in embryonic ptc-LacZ testes that expressed the Ptc receptor (Fig. 14). Cells that showed nuclear blue staining were situated in two loosely formed cellular layers immediately adjacent to and around the seminiferous cords. The cells in the layer closer to the cords appeared flattened and were presumed to be precursors to myoid cells. The outer layer consisted of cells that appeared rounder than those of the inner layer and were presumed to be early endothelial lymphatic cells. Although the nucleus of many of the cells in both of these layers stained blue, not all of these cells were stained (Fig. 14A). This pattern of staining was consistent around the periphery of the tubules throughout the testes. None of the cells in testes from wild-type embryos showed blue staining (Fig. 14B). As in the adult ptc-LacZ testis [8], there was no nuclear staining in presumed fetal Leydig cells located in the interstitial space; however, there was punctate blue staining in the cytoplasm of these cells in ptc-LacZ but not in wild-type embryos (not shown). Therefore, similar to our previous report [8], we were uncertain about expression of ptc in Leydig cells.
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DISCUSSION
The objective of this study was to address the primary defects that lead to the adult male phenotype observed in the testis of the Dhh-null mouse to understand more fully the function of Dhh in the male gonad. Study of developing, undifferentiated XY genital ridges and XY gonads from Dhh-null embryos revealed that normal production and/or deposition and full organization of the basal lamina that defined the embryonic seminiferous cords failed to occur. At 13.5 dpc, the embryos possessing at least one Dhh allele possessed a fully and continuously secreted basal lamina around the entire circumference of the embryonic seminiferous cords. Conversely, the Dhh-null embryos showed a defective basal lamina. The embryonic peritubular cells, contributors to the basal lamina, were identified as the targets of Dhh action by their expression of ptc. This observation supports the conclusion that the Dhh protein is a factor that plays an essential role in modulating the production and/or deposition of the basal lamina. All the defects verified in the Dhh-null mutant, from the embryonic formation of cords and the presence of germ cells outside of testicular cords through disorders in spermatogenesis by adulthood, are correlated with the defective organization of the basal lamina.
The basal lamina is a sheetlike extracellular structure in contact with the basal surface of all epithelial cells. It is composed of different extracellular matrix proteins, mainly collagen, laminin, and proteoglycans [17]. In the testis, the secretion of the basal lamina that surrounds the seminiferous tubules has been described as an event undertaken in cooperation between Sertoli cells and peritubular cells. In vitro cell culture and protein detection via immunologic probes have indicated that Sertoli cells contribute to the production and deposition of type IV collagen and small amounts of laminin, whereas peritubular cells release fibronectin and type I and type IV collagen [18].
The mechanism of action of the Dhh signaling pathway on the development of the basal lamina is unknown, as is how gaps appear in the basal lamina when Dhh is lacking. The absence of Dhh binding to its receptor at the level of the peritubular cells may be responsible for quantitatively insufficient activity of the cellular pathways that regulate production of those proteins that normally compose the basal lamina, or those proteins may be quantitatively adequate but the secretion itself may be abnormal at distinct points, which would induce the formation of a gap. The abnormal secretion hypothesis is supported by the fact that at 13.5 dpc in the Dhh-null mouse, the defective basal lamina not only showed gaps but also showed alternating regions of increased and reduced thickness, and by 16.5 dpc, interspersed thick patches of disorganized connective tissue were observed.
Pathologic variations in thickness of the basal lamina around the seminiferous tubules have been reported as features of cryptorchidism and/or presence of two X chromosomes [19–23]. Such alterations of the basal lamina are minor when compared with the serious abnormalities that accompany these conditions, which include cellular death and significant reduction of the vasculature. Therefore, it is difficult if not misleading to postulate any correlation between the features observed in the Dhh-null mouse and those described for cryptorchidism or chromosomal aberrations. In addition to these pathologic changes, the disruption of the basement membrane has also been linked to formation of certain carcinomas [24], which offers the possibility that a reduction or lack of testicular Dhh is involved in the formation of seminomas. However, the male and female Dhh-null mice of the study colony that have been kept for up to 2 yr have not developed tumors, testicular or otherwise. Thus, Dhh and its contribution to the deposition of basement membrane probably is not connected to the formation of cancerous testicular tumors.
In the Dhh-null mouse, germ cells have been found outside of seminiferous cords embryonically (present study) and neonatally [8]. The apparent passage of germ cells to the interstitium through gaps in the basal lamina by 13.5 dpc indicates that germ cells did come together correctly during the cellular organization that preceded formation of the cords and were thereby properly positioned by the time of deposition of the basal lamina. However, because the basal lamina was defective, physical communication between the interior cordal space and the interstitial space allowed a portion of the germ cells to move out of the cords and into the interstitium.
Although no systematic counting was undertaken in this study, Dhh-null gonads showed a relatively large number of germ cells outside the cords at 16.5 dpc and relatively fewer germ cells in the interstitium at 13.5 dpc. In XY mouse gonads, the mitotic activity of germ cells is suspended at 13.5 dpc and restarts only after birth [25]. Therefore, active migration of germ cells from inside the cordal space through the gaps in the basal lamina seems to be the best explanation for the apparent increase in number of germ cells present in the interstitium of Dhh-null gonads from 13.5 until 16.5 dpc. In addition, because germ cells were seen in the interstitium surrounded by undefined cell types, the movement of the germ cells may have been induced by a specific signal sent by cells in the interstitium.
In the present study, a structurally detectable defect was observed in the embryonic testis of mice lacking the Dhh gene, i.e., an irregular and gapped basal lamina that surrounds the developing seminiferous cords. Further studies are needed to explain how the inactivation of the Dhh signaling pathway leads to a failure of production or deposition of components of the basal lamina or improper organization of the deposited components into the final structure itself.
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ACKNOWLEDGMENTS
This paper is dedicated to Dr. Lonnie Russell, our colleague, mentor, and friend, who died while this paper was in press. He taught us more than he ever realized.
We are grateful to Angie Raymer and Ying Li for their help with tissue and processing of photographic material and to Dr. Blanche Capel for instruction in genital ridge dissection. We also thank Jean Flanagan, Sara Leiker, and Wendy Whoriskey for care of the animals and help in generating timed-pregnant female mice.
FOOTNOTES
First decision: 26 April 2001.
1 Supported by grant HD34594 to L.D.R. 
2 Correspondence and current address: Ann M. Clark, Serono Reproductive Biology Institute, 27 Pacella Park Dr., Randolph, MA 02368. FAX: 781 961 1043;ann.clark{at}serono.com.
3 Current address: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710.
Accepted: May 22, 2001.
Received: April 10, 2001.
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