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Research Article |
Prince Henry's Institute of Medical Research,3 Clayton Victoria, 3168, Australia
School of Engineering and Science,4 International University Bremen, Bremen 28758, Germany
Institute of Reproductive Medicine,5 University of Münster, Münster 48129, Germany
ABSTRACT
Sertoli cell number is considered to be stable and unmodifiable by hormones after puberty in mammals, although recent data using the seasonal breeding adult Djungarian hamster (Phodopus sungorus) model challenged this assertion by demonstrating a decrease in Sertoli cell number after gonadotropin depletion and a return to control levels following 7 days of FSH replacement. The present study aimed to determine whether adult Sertoli cells are terminally differentiated using known characteristics of cellular differentiation, including proliferation, junction protein localization, and expression of particular maturational markers, in the Djungarian hamster model. Adult long-day (LD) photoperiod (16L:8D) hamsters were exposed to short-day (SD) photoperiod (8L:16D) for 11 wk to suppress gonadotropins and then received exogenous FSH for up to 10 days. Sertoli cell proliferation was assessed by immunofluorescence by the colocalization of GATA4 and proliferating cell nuclear antigen and quantified by stereology. Markers of Sertoli cell maturation (immature, cytokeratin 18 [KRT18]; mature, GATA1) and junction proteins (actin, espin, claudin 11 [CLDN11], and tight junction protein 1 [TJP1, also known as ZO-1]) also were localized using confocal immunofluorescence. In response to FSH treatment, proliferation was upregulated within 2 days compared with SD controls (90% vs. 0.2%, P < 0.001) and declined gradually thereafter. In LD hamsters, junction proteins colocalized at the basal aspect of Sertoli cells, consistent with inter-Sertoli cell junctions, and were disordered within the Sertoli cell cytoplasm in SD animals. Exogenous FSH treatment promptly restored localization of these junction markers to the LD phenotype. Protein markers of maturity remain consistent with those of adult Sertoli cells. It is concluded that adult Sertoli cells are not terminally differentiated in the Djungarian hamster and that FSH plays an important role in governing the differentiation process. It is proposed that Sertoli cells can enter a transitional state, exhibiting features common to both undifferentiated and differentiated Sertoli cells.
seasonal reproduction, spermatogenesis, testis
INTRODUCTION
Numerous cell types are considered to be terminally differentiated in the adult animal, including neurons, myocytes, auditory hair cells, epidermal cells, and Sertoli cells [1, 2]. The Sertoli cell is a specialized testicular somatic cell that nurtures developing germ cells and is considered to be terminally differentiated after puberty [2]. Terminal differentiation involves loss of proliferative ability, formation of functional inter-Sertoli cell tight junctions, and expression of functions or proteins not present in immature Sertoli cells (for review, see [2]). Failure to undergo this differentiation process has been associated with infertility caused by testicular dysgenesis [3, 4]. The present study investigates the endocrine regulation of Sertoli cell differentiation in vivo.
The general view of a stable adult Sertoli cell population is largely derived from reports that division does not occur in normal [5] or hypophysectomized adult rats subsequently treated with hormones [6–9], nor do adult Sertoli cells degenerate after hypophysectomy [6, 7]. Sertoli cell numbers also do not change after chronic hormone suppression in the rat [10] and human [11]. A recent study used modern stereological techniques to demonstrate that Sertoli cell numbers were reduced by 34% in adult Djungarian hamsters exposed to short-day (SD) photoperiod [12], which results in suppressed serum gonadotropins, compared with numbers in long-day (LD) photoperiod controls [13]. Sertoli cell number in SD animals was normalized within 7 days of exogenous FSH replacement [12]. Fluctuations in Sertoli cell number in adult seasonal breeders also have been reported for the golden hamster [14], red deer [15], Soay ram [16], and stallion [17–19]. In further support that adult Sertoli cell numbers are capable of being altered, genetic manipulation of Sertoli cells from 20- and 60-day-old rats by overexpression of the helix-loop-helix inhibitor of differentiation proteins (ID1 and ID2) has demonstrated that adult Sertoli cells can reenter the cell cycle and proliferate in vitro [1].
The generally accepted view is that Sertoli cells proliferate during fetal and prepubertal life in mammals and are influenced by multiple endocrine and paracrine factors (for review, see [2]). Two key hormones that play a role in this process are FSH and thyroid hormone. Neonatal administration of FSH to rats increases the number of Sertoli cells [20, 21] by stimulating their proliferation [22]. Thyroid hormone regulates the cessation of Sertoli cell proliferation, because hypothyroid conditions extend the period of Sertoli cell proliferation [23, 24] and, thus, Sertoli cell number [25, 26]. In addition, treatment of Sertoli cells with thyroid hormone halts Sertoli cell proliferation in vitro in the rat [27] and in vivo in the mouse [24]. Testosterone is not thought to be important in dictating Sertoli cell proliferation, because normal numbers are observed in mice with a Sertoli cell-specific knockout of the androgen receptor gene [28].
Differentiation of Sertoli cells at puberty also coincides with the formation of the blood-testis barrier, which is comprised, in part, by membrane bound inter-Sertoli cell tight junction proteins and functions to provide an anatomical barrier between meiotic germ cells and most factors present in the general circulation [29]. Sertoli cell tight junctions contain three well-defined integral proteins, claudin 11 (CLDN11; also known as oligodendrocyte transmembrane/specific protein) [30], occludin [31, 32], and F11 receptor protein (also known as JAM-1) [33, 34], which are bound to intracellular plaque proteins, such as tight junction protein 1 (TJP1; also known as zona occludens 1) [35, 36] and the underlying actin cytoskeleton [36–38]. The CLDN11 is essential for fertility, because spermatogenesis in CLDN11-knockout animals is interrupted, with a complete loss of tight junction fibrils [39]. The TJP1 is associated with the cytoplasmic surface of tight junction proteins at sites of membrane fusion [36, 40, 41], but it also has been found to be associated with adherens junctions [40, 41] involved in Sertoli cell-germ cell adhesion and gap junctions [42] involved in intercellular communication. In the testis, tight junction proteins also are in close spatial association with the ectoplasmic specialization, which is a testis-specific, actin-based adherens junction [38, 43, 44] containing the actin-bundling protein espin [45].
The regulation of inter-Sertoli cell tight junctions is poorly understood, although the associated rise in serum gonadotropins and appearance of spermatocytes at puberty suggest endocrine and paracrine interactions [37, 46]. Tracer permeability studies have shown that the blood-testis barrier is nonfunctional in the SD Djungarian hamster and becomes functional shortly after recrudescence (the process of spermatogenic reactivation) is initiated [47], but to our knowledge, the regulation of tight junction and associated proteins in this species has not been studied.
In the Djungarian hamster, photoinhibition suppresses pituitary FSH and LH (and, as a result, testicular testosterone). As a consequence, spermatogenesis is disrupted primarily at the level of spermatogonial development [13, 47, 48]. Recrudescence in Djungarian hamsters occurs normally following restoration to an LD photoperiod and results in the normalization of serum gonadotropins [13] followed by restoration of the germ cell population [12]. Although relatively few studies have used this model to study the endocrine control of spermatogenesis, it is known that sperm production [13] and fertility [49] in the photoinhibited hamster are highly dependent on FSH. Testicular testosterone does not increase until 4 wk after recrudescence [50] and is reported to be necessary only for mounting behavior [13] in this species.
To account for the restoration of Sertoli cell number in the FSH-treated SD Djungarian hamster [12], we hypothesized that adult Sertoli cells are not terminally differentiated and must reenter the cell cycle and proliferate after FSH stimulation. The aims of the present study were to determine the differentiation status of Sertoli cells in hormonally manipulated adult hamsters as defined by 1) quantification of proliferative activity, 2) appearance and localization of junction and associated proteins (actin, espin, CLDN11, and TJP1) as markers of inter-Sertoli cell junction organization, and 3) expression of protein markers (KRT18 and GATA1) associated with Sertoli cell maturity. We demonstrate that FSH induces both proliferation of adult Sertoli cells and reorganization of inter-Sertoli cell junction proteins in SD animals.
MATERIALS AND METHODS
Animals
Thirty adult Djungarian hamsters (Phodopus sungorus) were bred, raised, and housed for 150 ± 30 days under artificially long photoperiods (16L:8D) and constant temperature (22°C) with free access to pelleted food and water in the colony of the Institute of Reproductive Medicine, University Münster (for details, see [51]). An additional group of five immature (Day 5) hamsters bred under LD photoperiod conditions from International University Bremen was included. All experiments were in accordance with local guidelines and with German law on the care and use of laboratory animals. All adult hamsters included in the experiments had large testes as determined by palpation at the onset of the experiment.
Experimental Design
Photoinhibition of reproductive function was induced by transfer of 30 adult hamsters from LD to SD photoperiods (8L:16D) for 12 wk, whereas one group of hamsters (n = 5) remained under LD photoperiods as reproductively active LD controls. The response to photoinhibition was assessed by palpation, after which all hamsters with no palpable testes were included in the study. Hamsters were then allocated to one of four groups (n = 5 per group), which received FSH treatment.
The FSH-treatment group animals received FSH (6 IU/day s.c.; recombinant human FSH; Gonal F Serono) reconstituted in sterile NaCl (0.154 M) daily for 2, 4, 6, and 10 days. Control animals received sterile NaCl (0.154 M). All animals were killed by decapitation under anesthesia. Testes were excised and weighed. The left testis was immersion-fixed in Bouin solution for 5 h. Tissues were sampled in a systematic, uniform, random approach as previously described [12]. Tissue was then dehydrated in graded ethanol washes (70%, 90%, and 100%), cleared in histolene (three times for 2 h each time), and embedded in paraffin wax before mounting on plastic backing blocks.
Immunohistology
Tissue sections (thickness, 5 µm) were cut and adhered to Superfrost-Plus slides (HD Scientific) before drying at 37°C for 1 h. Sections were dewaxed in histolene (twice for 8 min each time) and 100% ethanol (5 min) before air-drying and rehydration in graded ethanol (90%, 75%, and 50%) and, finally, in deionized H2O. Antigen retrieval was then performed by immersing sections in 600 ml of 1 mM EDTA-NaOH (pH 8.0) [52] and heating in an 800-W microwave oven set on high for two cycles of 5 min each before cooling for 1 h in EDTA buffer. Sections were then washed in deionized H2O followed by 10 mM PBS-1% BSA (5 min) before blocking in CAS-Block (Zymed) with 10% normal serum from the species in which the secondary antibody was raised for 20 min at room temperature. Primary antibodies were then applied as outlined in Table 1. Proliferating Sertoli cells were identified by the colocalization of GATA4 and proliferating cell nuclear antigen (PCNA), whereas GATA1 and KRT18 were used as markers of Sertoli cell maturity (Table 1). Sections were then washed in PBS-BSA (twice for 5 min each time) before addition of secondary antibody diluted 1:200 (goat anti-rabbit Alexa 488, goat anti-mouse Alexa 488 or Alexa 568, or rabbit anti-goat Alexa 488; Molecular Probes) and incubated at room temperature for 1 h. For dual immunohistology of proliferating Sertoli cells, sections were incubated in 10% normal goat serum (Chemicon International) for 20 min after secondary detection of GATA4 and before secondary detection of PCNA. Specificity of primary antibodies was verified by incubating sections in the equivalent concentration of antibody of the same isotype. Following secondary detection, all sections were washed in PBS-BSA (twice for 5 min each time) and dehydrated through graded ethanol (50%, 75%, 90%, and 100%) before brief immersion in histolene and air-drying. Sections were subsequently mounted in FluorSave (Calbiochem) under 22- x 50-mm coverslips (HD Scientific) and visualized using a confocal microscope (Fluoview FV300; Olympus Australia).
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Quantification of Proliferating Sertoli Cells
Both PCNA-positive and PCNA-negative Sertoli cells were quantified by stereology using a confocal microscope (final magnification, 600x). Fields were selected using a systematic, uniform approach from a random start, and images were collected. Between 100 and 300 Sertoli cells were counted for each hamster. An unbiased counting frame [12] was superimposed on each image, and cells were counted if they fell within the frame or touched the acceptance boundary. The percentage of PCNA-positive Sertoli cells was determined by dividing the total number of PCNA-labeled Sertoli cells by the total number of Sertoli cells (PCNA-positive and PCNA-negative). Sertoli cells were observed with varying intensities of PCNA staining and, therefore, were designated as PCNA-low, PCNA-intermediate, or PCNA-high. The PCNA-intermediate and PCNA-high Sertoli cells were included in the quantification of proliferating cells. Samples were run over seven experiments.
RESULTS
Sertoli Cell Proliferation
Qualitative assessment of GATA4 and PCNA dual immunohistology. In all groups tested, GATA4 reactivity in Sertoli cell nuclei was strong (Fig. 1, A–F). Reactivity with the GATA4 antibody also was identified in the acrosomes of elongating spermatids of the adult rat and LD hamster that was not observed in isotype control sections (Fig. 1, B and D). This is considered to be nonspecific staining, because no germ cell GATA4 reactivity has been identified in the adult testis of other rodent species.
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Sertoli cell nuclei reacted strongly with the PCNA antibody in the immature rat and immature hamster (Figs. 1, A and C). However, adult rat and adult LD hamster Sertoli cells demonstrated very little PCNA reactivity (Fig. 1, B and D), although a low level of expression was observed in some Sertoli cells (not shown). In the adult SD hamster, in which only Sertoli cells and spermatogonia are present within the cords [12, 14, 47], PCNA reactivity was restricted to germ cells (Fig. 1E) and a few isolated Sertoli cell nuclei (data not shown). In SD hamsters administered FSH (Fig. 1F), PCNA reactivity in Sertoli cells was markedly increased.
Quantitative assessment of FSH-stimulated Sertoli cell proliferation in SD hamsters. In response to FSH administration, Sertoli cell proliferation (as indicated by the colocalization of GATA4 and PCNA) was dramatically increased to 90% after 2 days, compared with 0.8% and 0.2% in adult LD and SD hamsters, respectively (both P < 0.001) (Fig. 2). Similar proliferative responses were observed in the 4-day (72%) and 10-day (68%) FSH treatment groups, although these responses were decreased compared with that in the 2-day treatment group (Fig. 2).
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Localization of Protein Markers of Sertoli Cell Maturation
GATA1 localization. The specificity of the GATA1 antibody was confirmed by immunostaining of mouse testis as a positive control, with staining localized in Sertoli cell nuclei (Fig. 3A) as previously observed [56, 57]. No GATA1 expression was identified in Sertoli cells of the immature hamster (Fig. 3B). In contrast to the situation in the mouse, GATA1 expression in the adult LD and SD hamsters was localized to the Sertoli cell cytoplasm (Figs. 3, C and D), an expression pattern unchanged by administration of exogenous FSH to the SD hamster (Fig. 3E). The cytoplasmic GATA1 staining pattern also was observed in the adult rat (data not shown).
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KRT18 localization. Expression of KRT18 was associated with Sertoli cells in the immature rat and hamster (Fig. 3, F and G), as shown by its localization in a filamentous pattern from the base of Sertoli cells. This expression pattern emulates that observed in other studies in rat [58] and human testis [62]. No staining for KRT18 was detected in the adult LD or SD hamster testis (Fig. 3, H and I), nor was it detected after FSH treatment of the SD animal (Fig. 3J).
Localization of Junction Proteins in the Testis
Actin and espin immunohistology. In the Day 5 hamster (Fig. 4A), actin expression within the seminiferous epithelium was weak and extended the entire depth of the cord. The intensity of espin staining also was weak and colocalized poorly with actin. In the adult LD hamster (Fig. 4B), actin expression localized to regions associated with inter-Sertoli cell junctions (along the basement membrane) and to Sertoli cell-spermatid junctions. Espin expression colocalized with actin at the basal aspect of Sertoli cells (Fig. 4B) and at Sertoli cell-spermatid junctions (Fig. 4B). In the adult SD hamster (Fig. 4C), actin staining was mainly restricted to remnants of the lumen, whereas weak espin immunoreactivity was found in Sertoli cell cytoplasm and did not colocalize with actin. In the SD hamster treated with FSH for 2 and 4 days (Fig. 4, D and E), actin and espin reactivity began to colocalize at the basal aspect of Sertoli cells and to extend through the tubule in radial processes. Following 10 days of FSH treatment (Fig. 4F), a greater proportion of actin and espin immunoreactivity was colocalized at the basement membrane relative to adluminal regions, although some reactivity was still apparent in adluminal regions.
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CLDN11 and TJP1 immunohistology. CLDN11 immunoreactivity was absent in cords of the immature hamster (Day 5) (Fig. 5A). Similarly, TJP1 staining was absent in the immature hamster testis (Fig. 6A), which is inconsistent with previous findings in the immature rat [41]. In the adult LD hamster (Fig. 5B), all CLDN11 reactivity was observed in a scalloped pattern parallel to the basement membrane, consistent with the area associated with inter-Sertoli cell tight junctions. A similar localization also was observed for TJP1 (Fig. 6B), although this protein was seen at Sertoli cell-spermatid junctions in some tubules as well (Fig. 6B). Espin colocalized extensively with CLDN11 and TJP1 at inter-Sertoli cell junctions (Figs. 5B and 6B). The intensity of both CLDN11 and TJP1 immunostaining also displayed some evidence of stage-specificity (Figs. 5B and 6B).
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In the adult SD hamster (Figs. 5C and 6C), CLDN11 and TJP1 localization was extensively disrupted and found in Sertoli cell cytoplasm. Tracer permeability studies have shown previously that tight junctions are nonfunctional in the SD animal and are functional in the LD animal [47]. Espin reactivity also was identified at a low level in the Sertoli cell cytoplasm, but colocalization with either CLDN11 or TJP1 was poor. After 2 days of FSH treatment (Figs. 5D and 6D), CLDN11 and TJP1 began to localize to basal aspects of Sertoli cells in association with espin. By 4 days, CLDN11 and TJP1 was extensively expressed at basal regions and colocalized with espin, with luminal espin staining persisting (Figs. 5E and 6E). After 10 days (Figs. 5F and 6F), strong CLDN11, TJP1, and espin reactivity was colocalized to basal aspects of Sertoli cells in closer association with the basement membrane than found at previous time points. This pattern of junction protein expression resembled the localization of these proteins in the LD animal (Fig. 5B and 6B). Similar effects of FSH on the localization of the tight junction protein occludin at the basal aspects of Sertoli cells in these adult tissues also were observed (data not shown).
DISCUSSION
The present study has demonstrated that adult Sertoli cells are not terminally differentiated in the photoinhibited Djungarian hamster, as evidenced by their ability to regain their proliferative activity after stimulation by exogenous FSH. To allow proliferation to occur, we hypothesized that adult LD Sertoli cells revert to an immature state in the SD setting, with features reflective of the prepubertal animal. This hypothesis is supported by the data in terms of proliferative ability and the pattern of tight junction protein localization, which returned to an LD phenotype. However, expression of established protein markers of Sertoli cell maturity was not supportive of the hypothesis, because we observed a failure of SD Sertoli cells to express KRT18 (typical of immature Sertoli cells) but a continued expression of GATA1 (a mature Sertoli cell marker). We therefore conclude that adult SD Sertoli cells are induced by FSH to transiently enter a proliferative state and to reorganize their junction protein localization while retaining some characteristics of mature Sertoli cells.
To our knowledge, the present study is the first to demonstrate the proliferation of Sertoli cells in an adult mammal in vivo, challenging the widely accepted view that adult Sertoli cells are terminally differentiated [2]. In support of this contention, a recent in vitro study has shown that adult rat Sertoli cells, which overexpress basic helix-loop-helix inhibitors of differentiation genes, reenter the cell cycle and proliferate [1]. It is well known that FSH plays a key role in supporting Sertoli cell proliferation in immature rodent models, but we now demonstrate that it has the same role in the adult Djungarian hamster.
The factors that drive reentry of Sertoli cells into the cell cycle are unknown, although in other models, both FSH and testosterone have been shown to play a role. Previously, FSH has been reported to induce stimulators of the cell cycle (e.g., cyclins [63]), whereas testosterone may indirectly influence Sertoli cell proliferation via induction of cell-cycle inhibitors (e.g., CDKN1B protein, also known as p27/Kip1) [24, 27]). In the SD Djungarian hamster, testicular androgens remain very low (4% of LD levels) during the first 4 wk of recrudescence [50], but FSH significantly increases within 3 days [13, 64]. Ablation of testosterone action by androgen-receptor antagonist treatment during FSH administration to SD Djungarian hamsters does not result in any differences in Sertoli and germ cell responses [12] compared to that with FSH treatment alone. We speculate that rising FSH in the presence of low testosterone generates the hormonal milieu that is conducive to Sertoli cell proliferation in an adult setting.
The majority of PCNA-positive Sertoli cells observed after administration of FSH suggests Sertoli cells in general are able to undergo proliferation rather than the existence of a population of Sertoli stem cells. However, the greater proportion of proliferating Sertoli cells than needed to account for the 34% increase observed after FSH treatment [12] suggests a mechanism (e.g., contact inhibition and/or apoptosis) that prevents overpopulation of Sertoli cells in the testis. It is noteworthy that a small proportion (<1%) of adult Sertoli cells in both LD and SD hamsters also was PCNA-positive. This could result either from the activation of DNA repair mechanisms (for review, see [59]) or from a small proportion of Sertoli cells being in cell cycle, a previously unreported finding.
Sertoli cells in the SD Djungarian hamster retain their adult phenotype as defined by two accepted immunohistochemical markers of Sertoli cell maturation (GATA1 [55, 57, 65] and KRT18 [62]). In the present study, GATA1 was localized to the cytoplasm of Djungarian hamster and rat Sertoli cells, in contrast to the mouse, in which GATA1 expression was restricted to the nucleus of Sertoli cells [55], highlighting a species-specific expression pattern of this transcription factor. In one model of Sertoli cell-specific knockout of the androgen receptor gene, no change was observed in the immunoexpression of markers associated with Sertoli cell maturation (including GATA1 and antimullerian hormone, also known as mullerian-inhibitory substance) [66], suggesting that androgens are not necessary for maturation-dependant changes in the expression pattern of these proteins.
The present study provides clear evidence that inter-Sertoli cell junction proteins are regulated by FSH, because the localization of protein markers of tight junctions and ectoplasmic specializations were disorganized in the SD animal, in which FSH is at or below the limits of assay detection [13] and is reorganized after FSH stimulation into a phenotype consistent with morphologically mature tight junctions at the basal aspect of seminiferous tubules, as seen in the LD hamster. Previously, FSH has been shown to regulate the organization of actin-containing junctions, such as the ectoplasmic specialization in vitro and in vivo [67, 68]. Similarly, actin filament organization across the first wave of spermatogenesis is coincident with increasing FSH levels while the testosterone level remains low [69, 70], as is the case in the early recrudescent Djungarian hamster [13]. This highlights the similarity between maturation of Sertoli cells at puberty and in the recrudescent hamster testis.
To our knowledge, the present study is the first in vivo demonstration of the ability of FSH alone to markedly alter the organization of the inter-Sertoli cell tight junction protein CLDN11, the tight and adherens junction-associated protein TJP1, and the ectoplasmic specialization-associated protein espin. In vitro studies have shown that both FSH and testosterone can upregulate rat Sertoli cell tight junction function [71], but a conflicting study found that FSH downregulates CLDN11 mRNA expression in mouse Sertoli cells in vitro [72]. A limited reduction in CLDN11 protein expression also was observed following selective knockout of the Sertoli cell androgen receptor [66], although this result was not replicated in a rat in vivo model of androgen depletion [34]. The disrupted organization of CLDN11, TJP1, and actin in the hamster correlates well with the known loss of blood-testis barrier functionality in the SD hamster [47]. Although not tested in the present study, we expect that functionality of the blood-testis barrier would have been restored (within 10 days) after FSH treatment, because localization of tight junction and associated proteins appeared to be similar to that in the LD hamster, in which a functional barrier is present [47]. This speculation is supported by the results of other studies that have demonstrated a correlation between organization of tight junction proteins as assessed by immunofluorescence microscopy and integrity of the blood-testis barrier as assessed using tracer techniques [34, 73]. In addition, 7 days of FSH treatment in this model were sufficient for the appearance of pachytene spermatocytes [12], which normally are seen only in tubules with a functional blood-testis barrier [44, 47].
In conclusion, the Djungarian hamster provides a unique natural model to identify the role of specific hormones and growth factors in the maintenance of adult Sertoli cell maturation. The present study demonstrates that adult Sertoli cells are not terminally differentiated, as previously considered, but are capable of reentering the cell cycle in this model. These Sertoli cells have characteristics of both adult and immature phenotypes, suggesting they take on an intermediate or transitional state. Elucidation of factors controlling Sertoli cell proliferation and junction organization could lead to insights regarding causative mechanisms driving testicular cancers and infertility syndromes.
ACKNOWLEDGMENTS
We gratefully acknowledge Dr. Stefan Schlatt (University of Pittsburgh School of Medicine, Pittsburgh, PA) for his guidance and support in establishing the hamster animal study, Karen Grote and Thomas Ströhlein from International University Bremen for their technical assistance, and Eberhard Nieschlag at University of Münster for support of the present study.
FOOTNOTES
1 Supported by the Wellcome Trust Fellow Scheme, U.K. 058479 (S.J.M.), and the National Health and Medical Research Council of Australia Program Grant 241000 (S.J.M. and P.G.S.) 
2 Correspondence: Sarah Meachem, Prince Henry's Institute of Medical Research, Monash Medical Centre, Block E, Level 4, 246 Clayton Road, Clayton, Victoria, 3168 Australia. FAX: 61 3 9594 6125; sarah.meachem{at}princehenrys.org
Received: 21 December 2005.
First decision: 4 January 2006.
Accepted: 5 January 2006.
REFERENCES
This article has been cited by other articles:
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  G. A Tarulli, S. J Meachem, S. Schlatt, and P. G Stanton Regulation of testicular tight junctions by gonadotrophins in the adult Djungarian hamster in vivo Reproduction, June 1, 2008; 135(6): 867 - 877. [Abstract] [Full Text] [PDF]
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 J.-B. Stukenborg, J. Wistuba, C. M. Luetjens, M. A. Elhija, M. Huleihel, E. Lunenfeld, J. Gromoll, E. Nieschlag, and S. Schlatt Coculture of Spermatogonia With Somatic Cells in a Novel Three-Dimensional Soft-Agar-Culture-System J Androl, May 1, 2008; 29(3): 312 - 329. [Abstract] [Full Text] [PDF]
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T. J Kaitu'u-Lino, P. Sluka, C. F H Foo, and P. G Stanton Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro Reproduction, June 1, 2007; 133(6): 1169 - 1179. [Abstract] [Full Text] [PDF]
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 S. J Meachem, S. Schlatt, S. M Ruwanpura, and P. G Stanton The effect of testosterone, dihydrotestosterone and oestradiol on the re-initiation of spermatogenesis in the adult photoinhibited Djungarian hamster J. Endocrinol., March 1, 2007; 192(3): 553 - 561. [Abstract] [Full Text] [PDF]
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