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Testis |
Pediatric Endocrinology Unit, Department of Woman and Child Health3
Karolinska Pharmacy,4 Karolinska Institute and Hospital, SE-171 76 Stockholm, Sweden
Departments of Pediatrics5 and
Anatomy, 6 University of Turku, FIN-20520 Turku, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, puberty, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Such germ cell apoptosis in the immature testis appears to be necessary for the normal maturation process associated with spermatogenesis, but to our knowledge, its physiological significance has not yet been elucidated in detail. For transgenic mice in which Bax, a proapoptotic member of the Bcl-2 family of proteins, has been "knocked out," the early wave of apoptosis is eliminated, resulting in the accumulation of spermatogonia and spermatocytes as well as infertility [5, 6]. Similar phenotypes have been observed in transgenic mice overexpressing Bcl-2 or Bcl-x, two antiapoptotic members of the Bcl-2 family [7, 8]. Thus, genetically induced alterations of the balance between apoptosis-protecting and apoptosis-inducing proteins in the testis are sufficient to disturb the normal development of functional spermatogenesis [6, 8].
The present investigation was designed to identify the cell types that are eliminated by apoptosis in the immature rat testis and to determine whether this apoptosis is associated with any particular stage of spermatogenesis. Type A and B spermatogonia, spermatocytes, and spermatids undergoing apoptosis exhibit a characteristic morphology that can be easily identified employing a phase-contrast microscope [9]. Therefore, supravital squash preparations were used to identify both the cell types undergoing apoptosis and the developmental phase of the germ cells present in sequential segments of the seminiferous tubules in rats at 8, 18, and 26 days of age. The findings thus obtained were confirmed by electron microscopy, TUNEL staining, and quantitation of the levels of activated caspase 3 and cleaved poly-(ADP-ribose)-polymerase (PARP) proteins. In addition, the levels of expression and localization (determined immunohistochemically) of the Bcl-2 proteins Bax, Bad, and Bcl-2 were compared to morphological findings.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats (BK-Universal, Stockholm, Sweden) at postnatal ages of 8, 18, and 26 days (eight animals per group) as well as 60 days (four animals per group) were housed at the animal facilities of Karolinska Hospital (Stockholm, Sweden) under an alternating 12L:12D photoperiod. The 8- and 18-day-old rats were placed in the same cages as their mothers, and the other animals were housed two in each cage. Free access to water and food was provided in all cases.
After killing the rats with CO2, the testes from four animals were removed. One testis from each animal was decapsulated, following which 1-mm sequential segments of a single long seminiferous tubule were prepared under a transillumination stereomicroscope in a Petri dish containing PBS (pH 7.4; 2 mM NaH2PO4, 8 mM Na2HPO4, and 150 mM NaCl). The immature seminiferous tubule exhibited no transillumination pattern comparable to that visualized in the adult testis [10]. The contralateral testis was frozen for protein analysis. From another four animals at postnatal ages of 8, 18, and 26 days, one testis was fixed for subsequent electron microscopy and the contralateral testis for immunohistochemistry (see below).
The present study was approved by the animal ethics committee of the Karolinska Institute (P.N. 169/97).
Viable Cell Preparations and In Situ 3'-End Labeling of DNA (TUNEL Analysis)
After isolation, each 1-mm tubular segment was transferred in 10 µl of PBS onto a microscope slide. After determining its diameter under the microscope, the segment was then carefully squashed under a coverslip. Squashing was monitored under a phase-contrast microscope and stopped when single cell layers were obtained.
The slides thus obtained were first examined under a phase-contrast microscope to identify the stages of the cycle, the most developed germ cell type, and apoptotic cells. Apoptosis of type A spermatogonia was seen to proceed in a manner similar to the corresponding process in somatic cells, with accumulation of heterochromatin followed by condensation of DNA at the periphery of the nucleus to form bright, phase-negative apoptotic bodies [9]. In the case of spermatocyte apoptosis, elevated amounts of heterochromatin were present just inside the nuclear envelope and deeper within the nucleus, giving rise to bright, phase-negative spheres [9].
The numbers of apoptotic cells per 1 mm of tubule length and per 1 µm3 of calculated volume were determined, and these values were confirmed by in situ end labeling of DNA strands (TUNEL analysis) in the same samples. After being rapidly frozen in liquid nitrogen and having the coverslip removed, the slides were dipped for a short period in ice-cold ethanol, fixed for 10 min in 4% formalin, washed twice with PBS for 5 min each time, postfixed in ethanol:acetic acid (2:1) at -20°C for 5 min, washed again with PBS as before, and finally dehydrated and stored at -70°C until analysis. The DNA 3'-end labeling was performed employing the standard procedure as described previously by Billig et al. [2]. Using distinct staining and an obviously apoptotic nucleus as criteria, the numbers of apoptotic cells per 1 mm of tubule length and 1 µm3 of calculated volume were quantitated.
Electron Microscopy
In preparation for electron-microscopic examination, testes were fixed by immersion in 5% glutaraldehyde in s-collidine buffer (0.16 M, pH 7.4) at 20°C. After 30 min of such treatment, the tissue samples were cut into approximately 1-mm3 cubes and thereafter reimmersed in the same fixative for an additional 2 h. Postfixation was performed with 1% osmium tetroxide in 1.5% aqueous potassium ferrocyanide, and the samples were then embedded in epoxy resin (Glycidether 100; Merck, Darmstadt Germany). Ultrathin sections (thickness, 70 nm) were prepared (Reichert E Ultramicrotome; Reichert Jung, Vienna, Austria), stained with uranyl acetate and lead citrate, and finally examined under a JEOL 100 SX electron microscope (JEOL, Tokyo, Japan).
Western Immunoblot Analysis
Total testicular protein was extracted employing modified RIPA buffer (1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 µg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM Na3VO4; and 1 mM NaF in 50 mM Tris-Cl, pH 7.4) and quantitated using the Bradford procedure (Bio-Rad, Hercules, CA). This protein fraction (30 µg of total protein for Bax, Bad, and PARP; 40 µg for caspase 3; and 60 µg for Bcl-2) from each animal was subjected to SDS-PAGE on 12% gels (8% in the case of PARP) under reducing conditions. Subsequently, the protein bands thus resolved were transferred electrophoretically to polyvinylidene fluoride membranes that had been preblocked overnight at 4°C in Tris-buffered saline (TBS; 150 mM NaCl in 10 mM Tris, pH 7.5) containing 5% nonfat dry milk.
These membranes were then incubated with primary rabbit polyclonal antibodies directed against Bad, Bax, caspase 3, and PARP (diluted 1:2000; Santa Cruz Biotechnology, Santa Cruz, CA) or against Bcl-2 (diluted 1:3500; Upstate Biotechnology, Lake Placid, NY) in TBS at room temperature for 1 h, washed five times with TBS containing 0.1% Tween-20 (PBS-T), and thereafter incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (diluted 1:104; Transduction Laboratories, San Diego, CA). The antigen-antibody complexes thus formed were quantitated by densitometry of the chemiluminescence emitted. After development of the films, the immunoblots were stained with Coomassie blue to confirm equal protein loading. Total testicular protein from each of the four rats in each group was immunoblotted in this manner.
Immunohistochemical Analysis
For immunohistochemical analysis, paraffin sections (thickness, 5 µm) of formalin-fixed testes were first dewaxed in xylene and subsequently rehydrated by immersion in a series of baths containing decreasing concentrations of ethanol. Antigen retrieval was achieved by incubation in 0.01 M sodium citrate (pH 6.0) at 95–98°C for 20 min, after which the slides were washed in PBS and endogenous peroxidase activity quenched by incubation with 3% H2O2 in PBS for 10 min. Nonspecific binding was blocked by incubation with 1% untreated goat serum for 1 h.
Subsequently, the slides were incubated with primary rabbit polyclonal antibodies directed against Bcl-2 (Upstate Biotechnology) or against Bax or Bad (Santa Cruz Biotechnology) at dilutions of 1:400, 1:200, and 1:200, respectively, in PBS containing 0.1% goat serum for 60 min. After extensive washing in PBS-T, these slides were then incubated for 30 min with a secondary biotinylated goat anti-rabbit immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:1000 in PBS containing 0.1% goat serum. After five additional washes with PBS-T, the sections were incubated with avidin-conjugated horseradish peroxidase (Vector Laboratories, Burlingame, CA). In the case of 18- and 26-day-old rats, adjacent sections were also stained with Mayer hematoxylin to allow accurate identification of the different stages of the seminiferous epithelial cycle. Sections of rat thymus and brain treated in this same manner served as positive controls. Negative-control sections were treated as described above, except that instead of being incubated with a primary antibody, normal serum from appropriate animal species was employed instead.
Statistical Analysis
In the case of vital cell preparations and TUNEL staining, at least 10 replicate samples from each of four rats in each group were examined. The corresponding number for the electron-microscopic investigation was three samples from each animal; for immunoblotting, the total protein fraction from each animal; and for immunohistochemical analysis, three samples from each animal. The quantitative data in the figures are presented as the mean ± SEM, whereas the nonparametric data in the table are presented as median values together with the 25% and 75% percentiles. The Mann-Whitney U-test was employed for comparison of independent groups of samples, and the Kruskall-Wallis analysis with the Dunn post-hoc test was performed for multiple comparison of independent groups of samples. A P value of less than 0.05 was considered to indicate a statistically significant difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At 8 days of postnatal age At this time-point, initiation of spermatogenesis could be observed. Large gonocytes exhibiting typical morphology and prominent nucleoli were seen to entering into mitosis (Fig. 1A). These gonocytes were surrounded by possible daughter cells, which demonstrated a size and morphology comparable to those of type A spermatogonia in the mature rat testis [9] (Fig. 1A). Neither intermediate nor type B spermatogonia were present. The Sertoli cells were relatively small and rounded-up, and some were undergoing mitosis. This morphological pattern was similar for each of the samples studied, and no individual stages of spermatogenesis could be identified.
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At 18 days of postnatal age At this point in development, pachytene spermatocytes in stage VIII were the most differentiated type of germ cell observed, although at a frequency lower than that in the 26-day-old rat testis. These samples could be divided into three groups on the basis of the presence of intermediate and type B spermatogonia in stages II–VI (Fig. 1B), preleptotene and midpachytene spermatocytes in stages VII–VIII (Fig. 1C), or type A spermatogonia, leptotene, and zygotene spermatocytes in stages IX–I (Fig. 1D). The exact numbers of samples in each group are presented in Table 1. Stages II–VI formed the shortest group of stages, having an average total length of 5 mm of the consecutive tubular segments. Spermatogonia in stages IX–I of the cycle exhibited few mitoses. The Sertoli cells were elongated, had nucleoli characteristic of mature Sertoli cells (Fig. 1B), and were never seen to be undergoing mitosis.
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At 26 days of postnatal age At this later time-point, meiotic divisions were observed, and step 3–4 spermatids were the most differentiated type of germ cell present (Fig. 1F). In comparison to the 18-day-old testis (Fig. 1C), the frequency of midpachytene spermatocytes at 26 days of age was increased (Fig. 1F), and the appearance of late pachytene spermatocytes and spermatids allowed accurate identification of the different stages of spermatogenesis. These samples were divided into three groups containing cells in stages II–VI, VII–VIII, or IX–I (Table 1). The characteristic cells observed were early pachytene and step 2–4 spermatids for stages II–VI (Fig. 1E), preleptotene and midpachytene spermatocytes for stages VII–VIII (Fig. 1F), and zygotene (Fig. 1G) and late pachytene spermatocytes and step 1, round spermatids for stages IX–I.
Morphological Characterization and TUNEL Staining of Apoptotic Germ Cells
At 8 days of postnatal age Type A spermatogonia and gonocytes were only rarely seen to undergo apoptosis. Apoptotic gonocytes were bigger than apoptotic spermatogonia, but accumulation of heterochromatin at the periphery of the nucleus was similar in both cases. No variation was observed in the number of apoptotic cells per 1 µm3 observed in different segments, nor was this value, as obtained by TUNEL staining, significantly different from that of the morphologic determination (Table 1).
At 18 days of postnatal age At this later phase, the frequency of cells demonstrating apoptotic condensation of the nucleus was greater than that in the 8-day-old testis (Table 1). The frequency of apoptosis among cells in stages IX–I was significantly higher than in the case of stages II–VI (Table 1). The apoptotic cells were identified as midpachytene spermatocytes (Fig. 1C) in stages VII–VIII and as type A spermatogonia in stages IX–I. In association with stages II–VI, only a few early pachytene spermatocytes were observed to undergo apoptosis, and no apoptotic intermediate or type B spermatogonia were present. In the case of pachytene spermatocytes, apoptotic condensation gave rise to bright, phase-negative spheres, a pattern clearly different from that seen with spermatogonia (Fig. 1C). These same apoptotic pachytene spermatocytes were shown to be TUNEL positive (Fig. 1C' and Table 1).
At 26 days of postnatal age In the 26-day-old rat testis, the number of apoptotic cells per 1 µm3 of tissue volume was also significantly higher than that in the 8-day-old testis (Table 1). On the other hand, the numbers of degenerating germ cells in the different stages of spermatogenesis per 1 µm3 of tissue volume were very similar to the corresponding values for 18-day-old testis (Table 1). At this older age, the numbers of apoptotic cells in stages IX–I and VII–VIII were significantly greater than the apoptotic frequency associated with stages II–VI (Table 1). The morphology of apoptotic type A spermatogonia in stages IX–I (Fig. 1G) and midpachytene spermatocytes in stages VII–VIII appeared to be identical to that of the corresponding cells in younger animals and were also shown to stain positively with the TUNEL procedure (Fig. 1, G and G', and Table 1). Intermediate and type B spermatogonia were not seen to undergo death by apoptosis.
At 60 days of postnatal age In the mature rat testis, the number of TUNEL-positive, apoptotic cells per 1 µm3 of tissue volume was lower than that observed in the immature 18- and 26-day-old testis (Table 1). The morphology of the different spermatogenic stages and stage-specific apoptosis in vital cell preparations derived from the mature rat testis have been described well in the literature [10, 11] and were therefore not examined here.
Electron-Microscopic Examination and Levels of Activated Caspase 3 and Cleaved PARP in the Maturing Rat Testis
At 8 days of postnatal age At this early time-point, abundant procaspase 3 (Fig. 2A), but low levels of activated caspase 3 (Fig. 2B) and cleaved PARP (Fig. 2C), were detected. Electron microscopic examination confirmed the apoptotic nature of spermatogonia and gonocyte death.
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At 18 and 26 days of postnatal age Electron-microscopic examination of the rat testis at these ages revealed degenerating spermatogonia in the vicinity of the basal membrane and nuclei with an appearance characteristic of apoptotic death. The only apoptotic cells associated with stages VII–VIII of the cycle were midpachytene spermatocytes. In general, the apoptotic midpachytene spermatocytes were stained more densely, and morphological features of the nucleus, including condensed chromatin cords, synaptonemal complexes, and sex vesicles, were still apparent (Fig. 3). The levels of activated caspase 3 (Fig. 2B) and cleaved PARP (Fig. 2C) peaked at the age of 18 days, thereafter declining with increasing age.
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Levels and Immunolocalization of Members of the Bcl-2 Family of Proteins in the Maturing Rat Testis
At 8 days of postnatal age This immature tissue expressed high levels of the prosurvival Bcl-2 protein (Fig. 4A), with the strongest immunohistochemical staining being exhibited by the gonocytes (Fig. 5A). Immunohistochemical staining for the Bax protein was also localized to the gonocytes and, in addition, the spermatogonia. In this case, the staining was particularly intense at the plasma membrane of large gonocytes in the vicinity of the basement membrane of the seminiferous tubule (Fig. 5B). The proapoptotic Bad protein was also expressed at a high level in the 8-day-old testis (Fig. 4C) and was localized immunohistochemically to the Sertoli cells (not shown).
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At 18 and 26 days of postnatal age At these later stages of testicular maturation, the levels of expression of the Bcl-2 protein has decreased considerably (Fig. 4A), and no immunohistochemically positive cells could be detected. In marked contrast, the proapoptotic Bax (Fig. 4B) and Bad (Fig. 4C) proteins demonstrated a peak of expression at these ages. Spermatogonia in contact with the basal membrane and in all stages of the spermatogenic cycle were stained with the anti-Bax antibody (Fig. 5, B–D). Furthermore, distinctly stage-dependent Bax staining of pachytene spermatocytes was observed in stages VII and VIII (Fig. 5, C and D). The most intense immunostaining of the Bad protein was associated with the Sertoli cells (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the immature rat testis, two groups of cells have been shown here to be especially prone to apoptosis. Type A spermatogonia were observed to die by apoptosis at 8 days of age, and furthermore, these cells accounted for a major proportion of the apoptotic cells in immature spermatogenic stages IX–I in older animals. This death of type A spermatogonia was associated with their maximal rate of mitosis in the same stages of the cycle; in addition, apoptotic A spermatogonia were surrounded by a large number of other, nonapoptotic A spermatogonia. These results support the earlier hypothesis that in the mammalian testis, the rate of degeneration of type A spermatogonia is density-dependent [12].
The high frequency of apoptosis during the immature spermatogenic stages VII and VIII was associated with midpachytene spermatocytes. In connection with these two stages, the frequency of apoptosis at 18 and 26 days of age was the same, whereas the total number of pachytene spermatocytes increased with increasing age, suggesting that cell density is not involved in regulating programmed cell death in this case. This pattern of elevated apoptosis in pachytene spermatocytes only during stages VII–VIII is identical to that observed after hypophysectomy [13]. In the latter case, lack of stimulation of Leydig cells by luteinizing hormone leads to a decrease in testosterone production, which in turn causes increased apoptosis in pachytene spermatocytes and spermatids at stage VII.
In the present study, the absence of haploid step 7–8 and elongated step 19 spermatids were the major features that distinguished spermatogenic stages VII and VIII in immature testis from the corresponding mature stages. In connection with pubertal spermatogenesis in the golden hamster, cessation of early spermatogenic apoptosis is known to coincide with the initiation of spermatid elongation [14]. During stages VII–VIII in the mouse, round spermatids secrete the paracrine factors Bmp8a and Bmp8b, which promote the survival of spermatocytes [15]. Accordingly, enhanced spermatocyte apoptosis is observed in mice in which the genes encoding these factors are defective [15]. Thus, in the immature testis, an absence of survival signals from round spermatids may contribute to the high frequency of apoptosis in spermatocytes.
Three members of the Bcl-2 family of proteins (i.e., Bcl-2 itself, Bax, and Bad) all exhibited age-dependent levels of expression in the rat testis. A high level of Bcl-2 expression, localized immunohistochemically to the gonocytes, was detected only in the youngest (i.e., 8-day-old) animals. Earlier studies have reported no significant expression of Bcl-2 in the rat or mouse testis [3, 8], despite the fact that in transgenic animals overexpressing this protein, a pronounced degenerative effect on spermatogenesis occurs [7, 8]. Our results here indicate that Bcl-2 is expressed at significant levels by rat gonocytes before their further maturation into spermatogonia. In contrast, testicular expression of Bax and Bad was found in the present study to peak at 18 and 26 days of age, which is in agreement with earlier observations of mice and rats [3, 8]. Employing immunohistochemical staining, Bad was localized primarily to Sertoli cells, confirming the earlier report of Yan et al. [3].
A novel finding of the present study was that the Bax protein exhibits a distinctly stage-dependent localization in immature rat testis. In earlier studies, Bax has been revealed immunohistochemically to be present in Sertoli cells, spermatogonia, and spermatocytes [3]. In the present investigation, intense expression of this proapoptotic protein was localized to pachytene spermatocytes in stages VII and VIII in 18- and 26-day-old rat testis. In addition, pronounced staining of gonocytes or spermatogonia, with no stage-dependency, was present at all ages. Because these groups of cells were also discovered to be particularly prone to apoptosis, it appears likely that in the immature testis, Bax regulates apoptosis in two distinct types of germ cells (i.e., the stem cells and midpachytene spermatocytes).
This conclusion is rather interesting in light of the disturbance in spermatogenesis associated with Bax. During the pubertal development of mice containing a targeted deletion of the antiapoptotic Bax, overproduction of spermatogonia, hyperplasia of the seminiferous tubule, and a block in the normal maturation of preleptotene spermatocytes are observed, resulting in later atrophy of the seminiferous epithelium [5, 6]. The midpachytene and preleptotene spermatocytes are nursed by the same Sertoli cell during spermatogenic stages VII–VIII (Fig. 3) in both the rat and mouse [16]. Thus, some correlation may exist between apoptosis in midpachytene spermatocytes and further maturation of preleptotene spermatocytes. The observation here that expression of Bax is localized to an advanced type of spermatocyte may explain why spermatogenesis in the Bax-deficient mouse can progress to such a relatively advanced state before permanent disruption of its organization and induction of Bax-independent apoptosis [5, 6].
The squash technique employed in the present study allowed rapid parallel detection of apoptotic cells together with accurate identification of the stage of spermatogenesis in immature rat seminiferous epithelium. The frequency of appearance of the different spermatogenic stages and the most advanced germ cell types in squashed tubular segments were comparable to earlier data from tubular cross-sections reported by Clermont and Perey [1]. When the kinetics of the appearance of the most advanced germ cells was compared to the data published by van Haaster and de Rooij [17], a similar accelerated rate of progression of the spermatogenic cycle during the first 18 days of life was observed, supporting the concept of different kinetics of immature and adult rat spermatogenesis [17]. Identification of unstained apoptotic cells on the basis of their morphology was as sensitive and reliable as TUNEL staining. The number of apoptotic cells was related to the volume of tissue (i.e., the length of the segment of seminiferous tubule), which enabled quantification of stage-specific apoptosis in the immature rat testis. Thus, our procedure offers the possibility of collecting viable testicular cells at different stages of spermatogenesis from immature animals for biochemical, physiological, and toxicological studies (e.g., for screening candidate drugs). In addition, it seems likely that this method could be further developed for clinical application to human testicular tissue.
In conclusion, we report here that as soon as the stages of spermatogenesis arise in the immature testis, apoptosis is stage-specific. Increased apoptosis of pachytene spermatocytes during stages VII and VIII represents the major difference between immature spermatogenesis and the corresponding adult process, and it is suggested to be an important regulatory aspect of the development of spermatogenesis in rats. Apoptosis during the first spermatogenic wave was mediated by activation of caspase 3 and was correlated to expression of members of the Bcl-2 family of proteins. Targeted apoptosis of midpachytene spermatocytes and stage-dependent, specific expression of the Bax protein in these same cells further indicate the important role played by this protein in maturation of the spermatogenic epithelium in the rat testis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Kirsi Jahnukainen, Department of Pediatrics, University of Turku, FIN-20520 Turku, Finland. Fax: 358 2 313 1460; kirsi.jahnukainen{at}kbh.ki.se
Received: 17 April 2003.
First decision: 11 May 2003.
Accepted: 23 September 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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