Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print July 7, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028183
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/5/1545    most recent
biolreprod.104.028183v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ahtiainen, M.
Right arrow Articles by Huhtaniemi, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ahtiainen, M.
Right arrow Articles by Huhtaniemi, I.
Agricola
Right arrow Articles by Ahtiainen, M.
Right arrow Articles by Huhtaniemi, I.
BIOLOGY OF REPRODUCTION 71, 1545–1550 (2004)
DOI: 10.1095/biolreprod.104.028183
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Indirect Sertoli Cell-Mediated Ablation of Germ Cells in Mice Expressing the Inhibin-{alpha} Promoter/Herpes Simplex Virus Thymidine Kinase Transgene1

Maarit Ahtiainen3, Jorma Toppari3, Matti Poutanen3, and Ilpo Huhtaniemi2,3,4

Department of Physiology,3 University of Turku, FIN-20520 Turku, Finland Institute of Reproductive and Developmental Biology,4 Imperial College, London W12 ONN, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we describe a novel mouse model for inducible germ cell ablation. The mice express herpes simplex virus thymidine kinase (HSV-TK) under the inhibin-{alpha} subunit promoter (Inh{alpha}). When adult transgenic (TG) mice were treated with famciclovir (FCV) for 4 wk, their spermatogenesis was totally abolished, with only Sertoli cells and few spermatids remaining in the seminiferous tubules. However, testicular steroidogenesis was not affected. Shorter treatment periods allowed us to follow up the progression of germ cell death: After 3 days, spermatogonia and preleptotene spermatocytes were no longer present. After a 1-wk treatment, spermatogonia, preleptotene, and zygotene spermatocytes were missing and the amount of pachytene spermatocytes was decreased. After a 2-wk treatment, round and elongating spermatids were present. During the third week, round spermatids were lost and, finally, after a 4-wk treatment, only Sertoli cells and few spermatids were present. Interestingly, the transgene is detected in Leydig and Sertoli cells but not in spermatogonia. This suggests that FCV is phosphorylated in Sertoli cells, and thereafter, leaks to neighboring spermatogonia, apparently through cell-cell junctions present, enabling trafficking of phosphorylated FCV. Because of the many mitotic divisions they pass through, the spermatogonia are very sensitive to toxins interfering with DNA replication, while nondividing Sertoli cells are protected. Using transillumination-assisted microdissection of the seminiferous tubules, the gene-expression patterns analyzed corresponded closely to the histologically observed progression of cell death. Thus, the model offers a new tool for studies on germ cell-Sertoli cell interactions by accurate alteration of the germ cell composition in seminiferous tubules.

bystander effect, germ cell ablation, germ cell-Sertoli cell interactions, HSV-TK, Sertoli cells, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The two main testicular functions are androgen production and spermatogenesis. The interstitial Leydig cells are responsible for testosterone (T) production, whereas spermatogenesis takes place in seminiferous tubules. Qualitatively and quantitatively normal spermatogenesis is strictly dependent on gonadotropins [1]. LH binds to its receptors located on Leydig cells, stimulating the production of T, which then diffuses from the interstitial compartment into seminiferous tubules. In turn, T binds to androgen receptors located in Sertoli and peritubular myoid cells and regulates, in this way, indirectly spermatogenesis [2]. FSH acts by binding to its receptors located on Sertoli cells and likewise controls spermatogenesis indirectly through regulation of Sertoli cell functions [3]. Besides these major regulators of spermatogenesis, an array of local paracrine factors produced by Leydig, peritubular, and germ cells modulate Sertoli cell functions and spermatogenesis [1, 4].

The seminiferous epithelium contains two types of cells: germ cells at different phases of maturation and the somatic Sertoli cells. The Sertoli cells, located in the basal part of the epithelium, are columnar in shape, reaching the whole thickness of the epithelium and surrounding the developing germ cells. Spermatogonia are located along the basement membrane of the seminiferous tubule, and spermatocytes and spermatids form the next layers toward the tubular lumen. According to morphologic features of the acrosome and the nuclei of developing spermatids, 12 stages of the seminiferous epithelial cycle can be distinguished in the mouse, and the duration of the cycle is 8.6 days [5]. In the mouse, it takes ~35 days for a single spermatogonium to complete spermatogenesis, as developing germ cells have to go through the cycle 4.5 times before being released into the tubular lumen at stage VIII of the cycle.

In the testis, Sertoli cells are the main site producing circulating inhibin B [6, 7]. Although Leydig cells express all inhibin subunit genes [8], it is controversial whether they produce also bioactive inhibin. We have earlier used inhibin {alpha}-promoter to direct the expression of an oncogene, SV 40 T-antigen, to gonads, thus producing Leydig and granulosa cell tumors [9, 10]. Using the same promoter, we produced another transgenic (TG) mouse line expressing herpes simplex virus thymidine kinase (Inh{alpha}/TK). By immunostaining, we showed the expression of TK both in Leydig and Sertoli cells [11]. When Inh{alpha}/Tag mice were crossbred with Inh{alpha}/TK mice and the double TG mice obtained were treated with ganciclovir, we were able to show the suitability of the model for testing gene therapy of gonadal tumors [11].

Herpes simplex virus thymidine kinase (HSV-TK) induces ablation of dividing cells upon antiherpes prodrug treatment. The viral enzyme monophosphorylates nucleoside analogues such as famciclovir (FCV), and the cytotoxic triphosphorylated product will incorporate into the elongating DNA of proliferating cells, resulting in termination of replication and cell death [12–14]. It has also been shown that neighboring cells not expressing HSV-TK can be killed by a bystander effect [15], apparently mediated through gap junctions present between adjacent cells [16 – 18]. However, the bystander effect has also been shown to occur without direct cell-cell contacts [19, 20]. In contrast with the sensitivity of proliferating Leydig tumor cells for the treatment [11], the present study shows that, in single TG Inh{alpha}/TK males, the most sensitive cells are located in seminiferous tubules. The data indicate that Leydig and Sertoli cells are highly resistant to the drug while germ cells are destroyed presumably by the bystander effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation and Identification of TG Mice

The TG mouse line used in this study expresses the HSV-TK coding sequence [21] under a 6-kilobase (kb) murine inhibin-{alpha} promoter sequence [9, 22] (Inh{alpha}/TK). Genotyping of the Inh{alpha}/TK transgene was performed on tail DNA of the offspring using a PCR method described previously [11]. The genetic background of the mice was FVB/N and age-matched wild-type (WT) littermates were used as controls.

After weaning at the age of 21 days, the mice were housed 4–6 per cage in conditions of controlled light (12L:12D) and temperature (21 ± 1°C). All mice were handled in accordance with the institutional animal care policies of the University of Turku (Turku, Finland), and the Animal Ethics Committee approved the studies.

FCV Treatments and Histopathological Analysis of Testes

At the age of 3 mo, the Inh{alpha}/TK males were treated with famciclovir (FCV; ~200 mg/kg body weight per day; SmithKline Beecham, West Sussex, UK; or FAMVIR 125 mg; SmithKline Beecham Pharmaceuticals, Crawley, UK) for different periods of time. FCV was added to drinking water (1 g/L) and the water was changed every second or third day. The given dose is estimated based on the water consumption and body weight of the mice.

To analyze the progression of cell ablation in seminiferous tubules, the mice were treated with FCV for 3 days, or 1, 2, 3, or 4 wk. After treatments, the mice were anesthetized with Avertin (tribromoethanol) [23], blood was collected by cardiac puncture, and the mice were then killed by cervical dislocation. Testis samples for histological analysis were fixed in freshly prepared 4% paraformaldehyde and embedded in paraffin. Five-micrometer sections were stained with hematoxylin-eosin.

To follow the time-course of putative recovery of spermatogenesis after FCV treatment, one testis was removed under anesthesia after 1-day, 3-day, or 1-wk FCV-treatment. Six weeks later, the mice were killed, and the remaining testis was taken for histological analysis.

Microdissection of Seminiferous Tubules and Real-Time Reverse Transcription-PCR

The mRNA expression of vimentin [24], inhibin-{alpha}, stem cell factor (SCF) [25], c-kit [25], and lactate dehydrogenase C (LDH C) [25] was analyzed by quantitative real-time reverse transcription (RT)-PCR. Untreated Inh{alpha}/TK mice and mice treated for 5 and 10 days were killed, their testes were removed and decapsulated, and the seminiferous tubules were microdissected. Tubule segments were isolated in Dulbecco modified Eagle F12 medium (DMEM/F12; Life Technologies, GIBCO BRL, Glasgow, UK) supplemented with 10 mg/L gentamycin sulphate and 1 g/L BSA under a stereomicroscope applying transillumination-assisted microdissection techniques as described previously (Fig. 1; [26]). Seven 2-mm-long pieces of each segment (S, spot-like [stages I–VI]; D, dark [stages VII– VIII]; P, pale [stages IX–XII]) were collected.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1. The 12 stages of mouse seminiferous epithelial cycle (top portion) beginning from type A4 spermatogonia (earlier types of spermatogonia are not shown, drawn according to de Rooij [55]) and the transillumination pattern of mouse seminiferous tubule (bottom portion). Arrows show the progression of cell death during various lengths of FCV treatments. In, Intermediate spermatogonia; B, type B spermatogonia; iPL, inactive preleptotene spermatocyte; aPL, active preleptotene spermatocyte; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; D, diplotene spermatocyte; Me, meiotic division; 1–16; 16 steps of spermiogenesis

Snap-frozen tissues were kept at –70°C until RNA isolation. Tissues were disrupted by passing them 10 times through a 19-gauge needle fitted to an RNase-free syringe and homogenized with QIGIAshredder Homogenizers (Qiagen, Valencia, CA). RNA extraction was made using RNeasy Mini kit (Qiagen) according to the manufacturer's protocol. Finally, RNA was eluted in 30 µl of RNase-free water.

The mRNA analyses were made by quantitative real-time RT-PCR by using the DNA Engine Opticon system (MJ Research, Inc., Waltham, MA) for continuous fluorescent detection. For each reaction, a 0.5-µl RNA sample was used and reactions were performed using QuantiTect SYBR Green RT-PCR Kit (Qiagen) according to the manufacturer's instructions. All samples and standards were amplified in triplicate. The expression level of ß-actin was used as reference to adjust for equal amount of sample RNA.

Hormone Measurements

T concentrations were measured by RIA from diethyl ether extracts of the sera and testis homogenates as described earlier [27]. Serum LH concentrations were measured by immunofluorometric assay for rat LH (Delfia; Wallac Oy, Turku, Finland) as described earlier [28].

Statistics

Statistical analyses were performed by using a SigmaStat program (version 2.0 for Windows 95; SPSS Inc., Chicago, IL). Kruskal-Wallis one-way analysis or one-way ANOVA was performed for analysis of statistical significance (P < 0.05). For pairwise multiple comparisons, the Student-Newman-Keuls test was used. All values are presented as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Leydig and Sertoli Cell Function

Our previous results have shown that the Inh{alpha}/TK TG is expressed in Leydig and Sertoli cells [11]. However, by treating the mice with FCV for different periods of time, the most profound change was the destruction of developing germ cells, whereas Sertoli and Leydig cells appeared resistant to the treatment. Normal function of Leydig cells was demonstrated by hormone measurements, as there was no significant difference in serum LH and T concentrations between Inh{alpha}/TK and WT males after the 4-wk FCV treatment. The LH concentrations were 0.38 ± 0.21 and 0.17 ± 0.10 µg/L, and those of testosterone 8.27 ± 5.51 and 8.09 ± 6.06 nmol/L in Inh{alpha}/TK and WT animals, respectively. Although the mean serum LH levels were clearly higher in Inh{alpha}/TK mice than in controls, the difference was not statistically significant because of high variation. Also, the serum T levels displayed large variation typical of male mice. Interestingly, T concentrations measured from testis homogenates were 2.7-fold higher in Inh{alpha}/TK males compared with WT males (55.2 ± 31.0 vs. 20.4 ± 23.9 pmol/ testis; P < 0.05).

Germ Cell Ablation

In contrast with Leydig and Sertoli cells, germ cells were markedly affected by the FCV treatment. After 3-days, spermatogonia and preleptotene spermatocytes were no longer detected (Fig. 2A), and after the 1-wk treatment, no spermatogonia, preleptotene, and zygotene spermatocytes were detected and the amount of pachytene spermatocytes was markedly decreased (Fig. 2B). After the 2-wk treatment, mostly round and elongating spermatids were present (Fig. 2C). During the third week of treatment, also round spermatids were lost (Fig. 2D), and finally, after the 4-wk FCV treatment, only Sertoli cells and few spermatids remained (Fig. 2E). FCV had no discernible effects on WT testes (Fig. 2F), demonstrating the absolute necessity of testicular TK expression for the prodrug activation. The progression of the detected cell ablation in the seminiferous epithelial cycle is shown in Figure 1. Destruction of seminiferous tubules was also reflected by the testis weights, which were 35.1 ± 2.9 mg in Inh{alpha}/TK males compared with 98.6 ± 7.2 mg in WT controls after the 4-wk treatment. The latter did not differ from nontreated WT males.



View larger version (114K):
[in this window]
[in a new window]
  		FIG. 2. Histological analyses of the testes of Inh{alpha}/TK males after a 3-day (A), a 1-wk (B), a 2-wk (C), a 3-wk (D), and a 4-wk (E) FCV treatment and of WT male after a 4-wk FCV treatment (F). The sections are stained with hematoxylin and eosin. In A–D, the arrows indicate the most immature spermatogenic cell type that can be observed after the treatment period in question. Pl, Preleptotene spermatocyte; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; RS, round spermatid; ES, elongating spermatid; SC, Sertoli cell; LC, Leydig cell (bar = 31 µm)

Effect of FCV Treatment on Tubular Gene Expression

The expression of genes specific for Sertoli cells (vimentin, inhibin-{alpha}, SCF), spermatogonia/spermatocytes (c-kit), and spermatids (LDH C) were analyzed by real-time RT-PCR in the testes 0, 5, and 10 days after initiation of the FCV treatment. The treatments did not affect the expression of vimentin and inhibin-{alpha} (Fig. 3, A and B). SCF expression was most abundant in stages I–VI and lowest in stages VII–VIII, where the expression was increased after the 5-day treatment (Fig. 3C). The expression of c-kit was decreased in each tubular segment analyzed, although the decrease was significant only in stages VII–VIII (Fig. 3D). Also, the expression of LDH C was decreased in stages VII–VIII after a 10-day FCV treatment (Fig. 3E).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 3. Quantitative RT-PCR analysis of vimentin, inhibin-{alpha}, SCF, c-kit, and LDH C in three different segments of the seminiferous tubule in untreated mice and in mice treated for 5 and 10 days with FCV. The bars show the relative expression of each gene compared with the expression of ß-actin mRNA. (n = 3 in each group; *, P < 0.05)

Assessment of Reversibility of the Germ Cell Death

We next studied whether the germ cell ablation was reversible after a short FCV treatment. This was done by treating the mice for 1 day, 3 days, and 1 wk (Fig. 4, A– C) and by allowing them to recover for 6 wk. Conspicuously, spermatogenesis did not recover after any of the treatments (Fig. 4, D–F). The data indicate that a 3-day treatment is sufficiently long to totally destroy the spermatogenesis (Fig. 4E), whereas some stages of the seminiferous epithelial cycle were able to escape the toxic effect of FCV during a 1-day treatment (Fig. 4D). This was indicated by complete spermatogenesis occasionally seen 6 wk after the treatment (Fig. 4D). Two possibilities for nonreversibility of FCV treatment exist; either all spermatogonial stem cells were destroyed during the treatment or the Sertoli cells were irreversibly damaged, being no more able to support the differentiation of germ cells. Three months after the 1-wk FCV treatment, obvious Leydig cell hypertrophy and hyperplasia were found, but no recovery of spermatogenesis (Fig. 4G).



View larger version (138K):
[in this window]
[in a new window]
  		FIG. 4. Histological analyses of the testes of Inh{alpha}/TK males 6 wk after a 1-day (A), a 3-day (B), and a 1-wk (C) FCV treatment (D, E, F, respectively) and 3 months after a 1-wk treatment (G). In A–C, the arrows indicate the most immature spermatogenic cell type that can be observed after the treatment period in question. Sd, Spermatid; Pl, preleptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; SC, Sertoli cell; LC, Leydig cell (bar = 31 µm)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we describe a novel gene-modified mouse model where germ cell ablation can be induced by FCV treatment. The model is based on testicular expression of the HSV-TK transgene, driven by a 6-kb-long murine inhibin-{alpha} promoter. In male mice, the promoter used directs the transgene expression into Leydig and Sertoli cells [11]. As the transgene is not expressed in any germinal cells, the prodrug FCV must be activated in Sertoli cells, expressing the transgenic TK activity, and then transported to the spermatogonia and spermatogonial stem cells surrounded by Sertoli cells. Spermatogonia go through several mitotic divisions, being therefore sensitive to toxins interfering with DNA replication. There is evidence that the cells located in the vicinity of HSV-TK-expressing cells can die because of the phenomenon called bystander effect [15]. Sertoli cells provide both structural and nutritional support for developing germ cells, and these cells are connected via several types of cell-cell junctions [29] offering trafficking routes for phosphorylated FCV from Sertoli cells to germ cells. Because the adult Sertoli cell population does not divide, it is protected from the toxic effect of FCV that is specific for cells with active DNA replication. The same apparently applies to the other cell type expressing TK in the current model, Leydig cells, which, in their functionally mature state, are terminally differentiated [30].

Suppression of spermatogenesis can be obtained in several ways. Exposure to heat degenerates primary spermatocytes and young spermatids [31]. In vitamin A deficiency mouse testis, the only remaining germ cells are the undifferentiated A spermatogonia [32]. Methoxy acetic acid, the active metabolite of a commonly used solvent, methoxyethanol, primarily afflicts primary spermatocytes [33]. In all these examples, spermatogenesis can be restored [31, 33, 34]. Similar to the FCV treatment of Inh{alpha}/TK mice, irradiation of testes selectively destroys premeiotic germ cells (differentiating spermatogonia), whereas somatic cells (Leydig and Sertoli) and postmeiotic germ cells (spermatids) are unaffected [5, 35–38]. Low radiation doses also allow repopulation of the destroyed cells in the seminiferous epithelium [35, 37]. Consequences of irradiation for the testes have been well described at the level of histology, but less is known about the possibility that the loss of germ cells of a certain stage could affect the other cells in terms of gene expression. It has been shown that apoptosis of radiation-sensitive cells causes a decrease in expression of factors specific for these cells (Fas, c-kit, and LIF-R), while the gene expression specific for radiation-resistant cells (SCF, Fas-L, TNF R55) were not affected [25]. The mRNA level for TGFß RI, which is expressed both in radiation-sensitive and resistant cells, decreased in radiation-sensitive cells but increased in resistant cells after irradiation.

In the present study, we analyzed the expression of genes specific for Sertoli cells (vimentin, inhibin-{alpha}, and SCF), spermatogonia/spermatocytes (c-kit), and spermatids (LDH C). In rats, the expression of inhibin-{alpha} was shown to increase in stages VII–VIII when pachytene spermatocytes and round spermatids were decreased in number [39]. In the present study, no difference in inhibin-{alpha} levels was found when we analyzed the gene expression after a 10-day FCV-treatment, at the time when round spermatids and part of the pachytene spermatocytes still existed in the seminiferous tubules. A very similar expression pattern for SCF in different stages of the seminiferous epithelial cycle was found previously in rats [40]. SCF expression was most abundant in stages I–VI and lowest in stages VII–VIII, where the expression was increased after 5-day FCV treatment. SCF has proven to be a survival factor for spermatogenic cells in rats [41], which might explain the increased expression as a response to declining germ cell population in the current study. Messenger RNA for the SCF receptor, c-kit, has been found in spermatogonia, primary spermatocytes, round spermatids, and Leydig cells [42–44]. The expression of c-kit was decreased in each tubular segment analyzed during the FCV treatment corresponding closely to the progressed cell death. LDH C, an enzyme for glucose metabolism, is exclusively present in postmeiotic germ cells [45]. For unknown reason, the expression of LDH C decreased in stages VII–VIII already after a 10-day FCV treatment preceding death of spermatids. Altogether, the expression pattern of the genes analyzed correlated closely with the progression of cell death.

It is known that atrophy of seminiferous tubules causes hypertrophy, increased content of cellular organelles, and increased steroidogenic capacity of Leydig cells [46–48]. It has been shown that the factors responsible for this response originate from Sertoli cells [49–51] and the secretion of this factor(s) is inhibited by pachytene spermatocytes [52]. This corresponds well to our results showing Leydig cell hypertrophy/hyperplasia and increased testicular T contents after the FCV treatment.

In our study, the germ cell ablation was irreversible after a short FCV treatment. From a cytological point of view, two types of A spermatogonia exist: the stem cells and the proliferating spermatogonia. The stem spermatogonia show no proliferating activity under normal circumstances [53]. These germ cells, however, undergo mitosis when the overall spermatogonial population is reduced, for example, after radiation [54]. Destruction of stem cells might explain why spermatogenesis does not recover after FCV treatment in the present study. The reduced number of dividing spermatogonia might stimulate stem spermatogonia to proliferate and become sensitive to FCV. It is still uncertain how long of a time the phosphorylated FCV stays in seminiferous tubules.

In conclusion, the current study provides a new model for studies on germ cell-Sertoli cell interactions in the mouse. Evidence exists that many functions of Sertoli cells are affected by interactions with germ cells. To understand the testicular phenotype in steadily increasing numbers of TG mouse models, and further, to understand human male infertility, these interactions need to be known. The currently described model offers a new tool for these purposes, as FCV treatments of different duration allow accurate alterations of the germ cell composition in seminiferous tubule and are also easy to allocate.


   ACKNOWLEDGMENTS
 
We thank Janne Suominen, MD, for microdisecting the seminiferous tubules. The skillful technical assistance of Ms. Nina Messner, Ms. Johanna Lahtinen, and Ms. Tarja Laiho is gratefully acknowledged.


   FOOTNOTES
 
1 Supported by grants from the Academy of Finland, Emil Aaltonen Foundation, and Sigrid Jusélius Foundation. Back

2 Correspondence: Ilpo Huhtaniemi, Institute of Reproductive and Developmental Biology, Imperial College, Faculty of Medicine, Du Cane Road, London W12 ONN, United Kingdom. FAX: 44 20 7594 2184; ilpo.huhtaniemi{at}imperial.ac.uk Back

Received: 5 February 2004.

First decision: 24 February 2004.

Accepted: 27 May 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Huhtaniemi I, Toppari H. Hormonal regulation of testis. In: Martínez-Garcia FRJ (ed.), Male Reproduction, a Multidisciplinary Overview. Spain: Churchill Communication; 1998:67–80
  2. Sar M, Hall SH, Wilson EM, French FS. Androgen regulation of Sertoli cells. In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL: Cache River Press; 1993:590–516
  3. Griswold MD. Actions of FSH on mammalian Sertoli cells. In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL: Cache River Press; 1993:493–508
  4. Jegou B. The Sertoli cell. Baillieres Clin Endocrinol Metab 1992 6:273-311[CrossRef][Medline]
  5. Oakberg EF. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Am J Anat 1956 99:507-516[CrossRef][Medline]
  6. Grootenhuis AJ, Timmerman MA, Hordijk PL, de Jong FH. Inhibin in immature rat Sertoli cell conditioned medium: a 32 kDa alpha beta-B dimer. Mol Cell Endocrinol 1990 70:109-116[CrossRef][Medline]
  7. Handelsman DJ, Spaliviero JA, Phippard AF. Highly vectorial secretion of inhibin by primate Sertoli cells in vitro. J Clin Endocrinol Metab 1990 71:1235-1238[Abstract]
  8. Roberts V, Meunier H, Sawchenko PE, Vale W. Differential production and regulation of inhibin subunits in rat testicular cell types. Endocrinology 1989 125:2350-2359[Abstract]
  9. Kananen K, Markkula M, Rainio E, Su JG, Hsueh AJ, Huhtaniemi IT. Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines. Mol Endocrinol 1995 9:616-627[Abstract]
  10. Kananen K, Markkula M, el-Hefnawy T, Zhang FP, Paukku T, Su JG, Hsueh AJ, Huhtaniemi I. The mouse inhibin alpha-subunit promoter directs SV40 T-antigen to Leydig cells in transgenic mice. Mol Cell Endocrinol 1996 119:135-146[CrossRef][Medline]
  11. Mikola MK, Rahman NA, Paukku TH, Ahtiainen PM, Vaskivuo TE, Tapanainen JS, Poutanen M, Huhtaniemi IT. Gonadal tumors of mice double transgenic for inhibin-alpha promoter-driven simian virus 40 T-antigen and herpes simplex virus thymidine kinase are sensitive to ganciclovir treatment. J Endocrinol 2001 170:79-90[Abstract]
  12. Nishiyama Y, Rapp F. Anticellular effects of 9-(2-hydroxyethoxymethyl) guanine against herpes simplex virus-transformed cells. J Gen Virol 1979 45:227-230[Abstract/Free Full Text]
  13. Furman PA, McGuirt PV, Keller PM, Fyfe JA, Elion GB. Inhibition by acyclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information. Virology 1980 102:420-430[CrossRef][Medline]
  14. Miller WH, Miller RL. Phosphorylation of acyclovir (acycloguanosine) monophosphate by GMP kinase. J Biol Chem 1980 255:7204-7207[Free Full Text]
  15. Freeman SM, Abboud CN, Whartenby KA, Packman CH, Koeplin DS, Moolten FL, Abraham GN. The "bystander effect:" tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 1993 53:5274-5283[Abstract/Free Full Text]
  16. Bi WL, Parysek LM, Warnick R, Stambrook PJ. In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy. Hum Gene Ther 1993 4:725-731[Medline]
  17. Yang L, Chiang Y, Lenz HJ, Danenberg KD, Spears CP, Gordon EM, Anderson WF, Parekh D. Intercellular communication mediates the bystander effect during herpes simplex thymidine kinase/ganciclovir-based gene therapy of human gastrointestinal tumor cells. Hum Gene Ther 1998 9:719-728[Medline]
  18. Carystinos GD, Katabi MM, Laird DW, Galipeau J, Chan H, Alaoui-Jamali MA, Batist G. Cyclic-AMP induction of gap junctional intercellular communication increases bystander effect in suicide gene therapy. Clin Cancer Res 1999 5:61-68[Abstract/Free Full Text]
  19. Bai S, Du L, Liu W, Whittle IR, He L. Tentative novel mechanism of the bystander effect in glioma gene therapy with HSV-TK/GCV system. Biochem Biophys Res Commun 1999 259:455-459[CrossRef][Medline]
  20. Princen F, Robe P, Lechanteur C, Mesnil M, Rigo JM, Gielen J, Merville MP, Bours V. A cell type-specific and gap junction-independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect. Clin Cancer Res 1999 5:3639-3644[Abstract/Free Full Text]
  21. McKnight SL. The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene. Nucleic Acids Res 1980 8:5949-5964[Abstract/Free Full Text]
  22. Su JG, Hsueh AJ. Characterization of mouse inhibin alpha gene and its promoter. Biochem Biophys Res Commun 1992 186:293-300[CrossRef][Medline]
  23. Hogan B, Beddington R, Constantini F, Lacy E. Manipulating the Mouse Embryo. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1994
  24. Ogino H, Satou W, Fujii M, Suzuki T, He Y, Michishita E, Ayusawa D. The human MYOD1 transgene is suppressed by 5-bromodeoxyuridine in mouse myoblasts. J Biochem (Tokyo) 2002 132:953-959[Abstract/Free Full Text]
  25. Mauduit C, Siah A, Foch M, Chapet O, Clippe S, Gerard JP, Benahmed M. Differential expression of growth factors in irradiated mouse testes. Int J Radiat Oncol Biol Phys 2001 50:203-212[CrossRef][Medline]
  26. Toppari J, Parvinen M. In vitro differentiation of rat seminiferous tubular segments from defined stages of the epithelial cycle morphologic and immunolocalization analysis. J Androl 1985 6:334-343[Abstract/Free Full Text]
  27. Huhtaniemi I, Nikula H, Rannikko S. Treatment of prostatic cancer with a gonadotropin-releasing hormone agonist analogue: acute and long term effects on endocrine functions of testis tissue. J Clin Endocrinol Metab 1985 61:698-704[Abstract]
  28. Haavisto AM, Pettersson K, Bergendahl M, Perheentupa A, Roser JF, Huhtaniemi I. A supersensitive immunofluorometric assay for rat luteinizing hormone. Endocrinology 1993 132:1687-1691[Abstract]
  29. Cheng CY, Mruk DD. Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development. Physiol Rev 2002 82:825-874[Abstract/Free Full Text]
  30. Moore A, Findlay K, Morris ID. In vitro DNA synthesis in Leydig and other interstitial cells of the rat testis. J Endocrinol 1992 134:247-255[Abstract]
  31. Jegou B, Laws AO, de Kretser DM. Changes in testicular function induced by short-term exposure of the rat testis to heat: further evidence for interaction of germ cells, Sertoli cells and Leydig cells. Int J Androl 1984 7:244-257[Medline]
  32. van Pelt AM, de Rooij DG. Synchronization of the seminiferous epithelium after vitamin A replacement in vitamin A-deficient mice. Biol Reprod 1990 43:363-367[Abstract]
  33. Bartlett JM, Kerr JB, Sharpe RM. The selective removal of pachytene spermatocytes using methoxy acetic acid as an approach to the study in vivo of paracrine interactions in the testis. J Androl 1988 9:31-40[Abstract/Free Full Text]
  34. van Pelt AM, de Rooij DG. Retinoic acid is able to reinitiate spermatogenesis in vitamin A-deficient rats and high replicate doses support the full development of spermatogenic cells. Endocrinology 1991 128:697-704[Abstract]
  35. van Beek ME, Davids JA, de Rooij DG. Variation in the sensitivity of the mouse spermatogonial stem cell population to fission neutron irradiation during the cycle of the seminiferous epithelium. Radiat Res 1986 108:282-295[CrossRef][Medline]
  36. Joshi DS, Yick J, Murray D, Meistrich ML. Stage-dependent variation in the radiosensitivity of DNA in developing male germ cells. Radiat Res 1990 121:274-281[Medline]
  37. van der Meer Y, Huiskamp R, Davids JA, van der Tweel I, de Rooij DG. The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation. Radiat Res 1992 130:289-295[CrossRef][Medline]
  38. Vergouwen RP, Huiskamp R, Bas RJ, Roepers-Gajadien HL, de Jong FH, van Eerdenburg FJ, Davids JA, de Rooij DG. Radiosensitivity of testicular cells in the prepubertal mouse. Radiat Res 1994 139:316-326[CrossRef][Medline]
  39. Kaipia A, Parvinen M, Shimasaki S, Ling N, Toppari J. Stage-specific cellular regulation of inhibin alpha-subunit mRNA expression in the rat seminiferous epithelium. Mol Cell Endocrinol 1991 82:165-173[CrossRef][Medline]
  40. Hakovirta H, Yan W, Kaleva M, Zhang F, Vanttinen K, Morris PL, Soder M, Parvinen M, Toppari J. Function of stem cell factor as a survival factor of spermatogonia and localization of messenger ribonucleic acid in the rat seminiferous epithelium. Endocrinology 1999 140:1492-1498[Abstract/Free Full Text]
  41. Yan W, Suominen J, Samson M, Jegou B, Toppari J. Involvement of Bcl-2 family proteins in germ cell apoptosis during testicular development in the rat and pro-survival effect of stem cell factor on germ cells in vitro. Mol Cell Endocrinol 2000 165:115-129[CrossRef][Medline]
  42. Manova K, Nocka K, Besmer P, Bachvarova RF. Gonadal expression of c-kit encoded at the W locus of the mouse. Development 1990 110:1057-1069[Abstract/Free Full Text]
  43. Manova K, Huang EJ, Angeles M, De Leon V, Sanchez S, Pronovost SM, Besmer P, Bachvarova RF. The expression pattern of the c-kit ligand in gonads of mice supports a role for the c-kit receptor in oocyte growth and in proliferation of spermatogonia. Dev Biol 1993 157:85-99[CrossRef][Medline]
  44. Rossi P, Marziali G, Albanesi C, Charlesworth A, Geremia R, Sorrentino V. A novel c-kit transcript, potentially encoding a truncated receptor, originates within a kit gene intron in mouse spermatids. Dev Biol 1992 152:203-207[CrossRef][Medline]
  45. Cooker LA, Brooke CD, Kumari M, Hofmann MC, Millan JL, Goldberg E. Genomic structure and promoter activity of the human testis lactate dehydrogenase gene. Biol Reprod 1993 48:1309-1319[Abstract]
  46. Aoki A, Fawcett DW. Is there a local feedback from the seminiferous tubules affecting activity of the Leydig cells?. Biol Reprod 1978 19:144-158[Abstract]
  47. de Kretser DM. Local regulation of testicular function. Int Rev Cytol 1987 109:89-112[Medline]
  48. Saez JM, Perrard-Sapori MH, Chatelain PG, Tabone E, Rivarola MA. Paracrine regulation of testicular function. J Steroid Biochem 1987 27:317-329[CrossRef][Medline]
  49. Janecki A, Jakubowiak A, Lukaszyk A. Stimulatory effect of Sertoli cell secretory products on testosterone secretion by purified Leydig cells in primary culture. Mol Cell Endocrinol 1985 42:235-243[CrossRef][Medline]
  50. Verhoeven G, Cailleau J. Specificity and partial purification of a factor in spent media from Sertoli cell-enriched cultures that stimulates steroidogenesis in Leydig cells. J Steroid Biochem 1986 25:393-402[CrossRef][Medline]
  51. Verhoeven G, Cailleau J. Influence of coculture with Sertoli cells on steroidogenesis in immature rat Leydig cells. Mol Cell Endocrinol 1990 71:239-251[CrossRef][Medline]
  52. Onoda M, Djakiew D, Papadopoulos V. Pachytene spermatocytes regulate the secretion of Sertoli cell protein(s) which stimulate Leydig cell steroidogenesis. Mol Cell Endocrinol 1991 77:207-216[CrossRef][Medline]
  53. Schlatt S, Weinbauer GF. Immunohistochemical localization of proliferating cell nuclear antigen as a tool to study cell proliferation in rodent and primate testes. Int J Androl 1994 17:214-222[Medline]
  54. Meistrich M, van Beek MEAB. Spermatogonial stem cells. In: Desjardins C, Ewing LL (eds.), Cell and Molecular Biology of the Testis. Oxford: Oxford University Press; 1993:266–295
  55. De Rooij DG. Stem cells in the testis. Int J Exp Path 1998 79:67-80[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/5/1545    most recent
biolreprod.104.028183v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ahtiainen, M.
Right arrow Articles by Huhtaniemi, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ahtiainen, M.
Right arrow Articles by Huhtaniemi, I.
Agricola
Right arrow Articles by Ahtiainen, M.
Right arrow Articles by Huhtaniemi, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS