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Endocrinology Vol. 140, No. 6 2746-2754
Copyright © 1999 by The Endocrine Society

ARTICLES

Germ Cell-Specific Cyclic Adenosine 3',5'-Monophosphate Response Element Modulator Expression in Rodent and Primate Testis Is Maintained Despite Gonadotropin Deficiency1

Rüdiger Behr and Gerhard F. Weinbauer

Institute of Reproductive Medicine of the University, D-48129 Munster, Germany

Address all correspondence and requests for reprints to: Prof. Dr. Gerhard F. Weinbauer, Institute of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Munster, Germany. E-mail: weinbau{at}uni-muenster.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
cAMP response element modulator (CREM) is an important component of the cAMP-mediated signaling pathway and is essential for differentiation of haploid male germ cells. In the rodent, testicular expression of CREM is believed to be controlled by FSH. We studied the expression pattern of CREM and gonadotropic control in the nonhuman primate and rodent testis. Adult cynomolgus monkeys (Macaca fascicularis) received daily either vehicle or the potent GnRH antagonist (ANT) cetrorelix for periods of 25 and 56 days. Rats were also exposed to vehicle or ANT for periods of 14 and 42 days. ANT treatment suppressed pituitary gonadotropin secretion, reduced testis size, and altered spermatogenesis. A rabbit polyclonal antibody raised against recombinant CREM{tau} and reacting with CREM{alpha}, -ß, -{gamma}, -{tau}1, and -{tau}2 at similar affinities was used for immunocytochemistry and Western blotting. CREM expression was seen in round spermatids, with highest levels during spermatogenic stages V–VII, but declined with progression of spermatid development in the primate. Similar observations were made for the rat testis. Thus, CREM expression was maximal at the onset of acrosome formation and was low or undetectable upon initiation of spermatid elongation in both species. A weak, but specific, CREM signal was seen in mid- to late pachytene spermatocytes and during meiotic division in both species. After ANT exposure, the germ cell- and stage-specific pattern of CREM expression was quantitatively retained at all time points and in both species. Northern and Western blot analysis confirmed the maintenance of testicular CREM expression despite 25 days of ANT treatment. A retrospective immunocytochemical analysis of rat testes 14 days posthypophysectomy revealed CREM signals in round spermatids. These findings demonstrate that the testicular expression of CREM is not entirely dependent on gonadotropic hormones but, rather, on the maturational stage of haploid round germ cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SPERMATOGENESIS is a particularly complex process encompassing the production of haploid mature spermatozoa from diploid spermatogonial stem cells. This process also comprises differentiation and morphogenesis of the haploid germ cells (spermiogenesis). Quantitatively normal production and maturation of haploid spermatids in the seminiferous epithelium depends on the gonadotropic hormones FSH and LH, which are released in response to hypothalamic GnRH (1, 2). The relative importance of each gonadotropin, however, is still under debate (3, 4, 5, 6). The cellular targets of FSH and LH are the Sertoli cell and the Leydig cell, respectively. Gonadotropic hormones increase intracellular cAMP levels via the adenylyl cyclase-coupled receptor (7, 8) followed by activation of protein kinase A (9). Subunits of cAMP-dependent kinases are expressed in Leydig cells, Sertoli cells, and germ cells (10). Among the various substrates for protein kinase A are cAMP-response element (CRE)-binding protein (CREB) (11) and CRE modulator (CREM) (12, 13). Alternative splicing and alternative promoter usage events generate activators and repressors of CREB and CREM (12, 13, 14), and upon phosphorylation, these transcription factors bind to CREs and modulate the transcriptional activity of cAMP-responsive genes (15). CREB and CREM are expressed in somatic and some germinal cells of the testis (14, 16, 17).

Male mice lacking functional CREM proteins were infertile, with spermiogenesis arrested at the level of round spermatids (18, 19). The CREM gene is differentially regulated during spermatogenesis: repressors ({alpha}, ß, and {gamma}) are expressed in premeiotic and early meiotic germ cells, whereas activators ({tau}, {tau}1, and {tau}2) are abundant in postmeiotic germ cells (20). Low expression of CREM{tau} activator has also been found in pachytene spermatocytes (21). The developmental switch from repressor to activator has been reported to be FSH dependent in rats and golden hamsters and is thought to be mediated via increased CREM activator transcript stability (22, 23). A CREM repressor lacking the trans-activation domain has been described in rat elongated spermatids (24). Moreover, expression of a truncated CREM isoform, inducible cAMP early repressor, that arises from the use of an alternative promoter has been described for Sertoli cells (25). The cellular distribution of CREM proteins has been reported in the mouse testis using an antiserum raised against CREM{tau} (21). Expression signals were confined to round spermatids from stage IV onward, with highest expression levels during stages VII and VIII. Recently, we described the localization of CREM protein in round spermatids and in pachytene spermatocytes and some Sertoli cells in men (26). The present work investigates the pattern and gonadotropin control of testicular CREM expression in nonhuman primates and rats. GnRH antagonist (ANT) was administered, as ANT are known to effectively suppress gonadotropic hormone secretion in rats and primates (2, 27, 28). We observed germ cell- and stage-specific CREM expression. However, testicular expression of CREM was retained despite gonadotropin deficiency, but was related to the developmental stage of haploid germ cell maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult Sprague Dawley rats (300–400 g body weight) and cynomolgus monkeys (Macaca fasicularis; 4.4–7.0 kg BW) were maintained under a 12-h day, 12-h night regimen in a temperature-controlled environment. Animals had access to species-specific pelleted food and unlimited access to tap water, and monkey chow was supplemented daily with fresh fruit. All experimental studies were undertaken in accordance with the German Federal Law on the Care and Use of Laboratory Animals.

Experimental studies
Study 1. Monkeys received daily sc injections of ANT (cetrorelix; 450 µg/kg) or vehicle (VEH; 5.25% glucose in physiological saline; n = 5/group) for 25 days. Testis tissue was collected after 16 and 25 days in the course of an investigation on spermatogenesis (29, 30). Tissue sampled on day 25 was used for immunocytochemistry, and parts were snap-frozen for Northern and Western blot analysis of CREM expression. Blood was collected from the cubital vein, and testicular volumes were measured using calipers (31). Serum testosterone (T) concentrations, reflecting LH secretion, were reduced about 10-fold by ANT and were close to the range for orchidectomized animals (<2 nmol/liter) after 25 days of ANT. Serum inhibin levels, reflecting FSH secretion, were close to the detection limit of the assay. Testicular volume and number of round spermatids were reduced by about 50%.

Study 2. To evaluate the effects of longer term gonadotropin withdrawal on CREM expression in the primate, monkeys were treated with ANT or VEH (n = 5/group) as described above, but over a period of 56 days. Testicular volumes, sperm numbers in the ejaculate, and serum T levels were determined weekly throughout the study. Ejaculates were collected and analyzed as described previously (31). Testicular biopsies obtained on day 56 were used for immunocytochemistry. Serum T concentrations ranged between 24 ± 6 and 44 ± 6 nmol/liter in the VEH group. In the ANT group, T levels were reduced from 51 ± 9 to below 5 nmol/liter within 2 weeks and to 1.6 ± 0.1 nmol/liter on day 56. Inhibin levels were not determined. Testicular volume (left plus right) was, on the average, 20–24 ml in the VEH group. Under ANT, testis dimensions were 25 ± 1 ml at baseline, declined steadily, and had shrunken to 11 ± 1 ml by 8 weeks (P < 0.05). In the ANT group, baseline sperm numbers were 45–72 x 106/ejaculate and dropped to 0 between weeks 7–8 of treatment, but remained unchanged in the VEH group.

Study 3. Rats received sc injections of ANT (450 µg/kg) or VEH for 14 days (n = 5/group). At death, trunk blood was collected, and testes were weighed and prepared for immunocytochemistry and Northern and Western blot analyses of CREM expression. Two additional groups were maintained on VEH or ANT treatment for 42 days (n = 4/group), and testicular tissue was prepared for immunocytochemistry. Serum T and FSH levels were 7.6 ± 1.7 nmol/liter and 5.5 ± 0.3 µg/liter in VEH groups, respectively, whereas hormone levels were below the assay detection limits by day 14 of ANT. Testes weights were 1.7 ± 0.1 (VEH) vs. 1.3 ± 0.1 g (ANT; P < 0.05) after 14 days and 1.8 ± 0.05 vs. 0.3 ± 0.002 g (P < 0.05) after 42 days.

Study 4. CREM immunolocalization was performed on rat testes (n = 4/group) 14 days after hypophysectomy alone or supplemented with human FSH treatment (two doses of 5 IU/day). The endocrine and spermatogenic status has been reported previously (32). For animals not supplemented with hormones, testes weight was reduced by 70%, FSH concentrations were at the assay detection limit, and T levels were reduced to below 1 nmol/liter. No pituitary remnants were found at autopsy.

Immunocytochemistry
Bouin’s-fixed and paraffin-embedded specimens were sectioned at 5 µm (26). Initially, an antigen retrieval step was performed by microwaving the sections in 0.05 M glycine buffer for 20 min (Bio-Rad H2500 microwave processor, Bio-Rad Laboratories, Inc., Richmond, CA). A rabbit polyclonal antibody raised against recombinant CREM{tau} (Upstate Biotechnology, Inc., Lake Placid, NY) and recognizing CREM{alpha}, -ß, -{gamma}, -{tau}1, and -{tau}2 with similar affinity, was used at 1:400 to 1:1200 dilutions. Five percent normal porcine serum was used to avoid nonspecific binding. Biotinylated antirabbit IgG from swine (1:400; DAKO Corp., Hamburg, Germany), extravidin-conjugated alkaline phosphatase (1:200; Sigma Chemical Co., St. Louis, MO), and New-Fuchsin (DAKO Corp.) were employed for detection of bound primary antibody. Mayer’s hematoxylin was used as counterstain. The method and primary antibody specificity were assessed by replacement with 1% (wt/vol) BSA or preincubation with recombinant human CREM. Stage classification of spermatogenesis was performed as described for the rat (33, 34) and macaque (35, 36). CREM staining was analyzed by image analysis (KS 400, Carl Zeiss Jena GmbH, Oberkochen, Germany) as described recently (26). Identification of CREM-positive cells was based on the interactive mode threshold setting. For each positive cell, the integrated signal across the entire cell was measured automatically by a built-in densitometry algorithm. As our system is not equipped with calibration software, measurements were expressed in arbitrary units of the equipment-specific gray scale. Fifty to 200 cells were analyzed per stage and animal.

Western blot analysis
Testicular tissue was homogenized in RIPA buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the protein concentration was determined by bicinchoninic assay (26). Four hundred micrograms of protein from testicular extract were loaded onto 10% PAGE and blotted onto nitrocellulose membrane. The blot was probed with CREM antibody at a dilution of 1:500, second antibody was used at 1:3000, and bands were detected by enhanced chemiluminescence. Unspecific binding was blocked with 0.5% Tween-20 and 5% nonfat milk powder. Full-length polyhistidine-tagged human CREM was used as a positive control.

Northern blot analysis
Tissue was homogenized using RNAzol B (Biotex Laboratories, Inc., Houston, TX), followed by chloroform extraction of RNA, precipitation with isopropanol at 4 C for 2–3 h, washes in 75% ethanol, and dilution in diethylpyrocarbonate-water. Samples were electrophoresed in 1% agarose/1 x N-morpholino-3-propane-sulfonic acid/formaldehyde gels, blotted onto nylon membranes (Amersham, Braunschweig, Germany), and fixed by baking at 80 C for 2 h or cross-linked by UV irradiation. Filters were prehybridized at 50 C for 60 min in digoxigenin Easy Hyb buffer (Boehringer Mannheim, Mannheim, Germany). Hybridization conditions were identical to those of prehybridization with the addition of the digoxigenin-labeled complementary DNA probe to a final concentration of 10–20 ng/ml hybridization solution. The CREM complementary DNA probe comprised exons E, F, G, H, and Ib (17) of the monkey. Hybridizations were performed at 50 C for 16 h. Filters were washed twice for 5 min each time in 2 x SSC (standard saline citrate)-0.1% SDS at room temperature and twice for 15 min each time in 0.2 x SSC-0.1% SDS at 50 C, and the digoxigenin labeling and detection kit (Boehringer Mannheim) was used for visualization. Filters were exposed to Amersham High Performance films for 2–4 h. For the rat testis, hybridizations (16 h at 66 C; Express Hyb buffer, CLONTECH Laboratories, Inc., Palo Alto, CA) were also performed using 32P-labeled probes (final concentration, 1 x 106 cpm/ml; HighPrime, Boehringer Mannheim). Filters were washed four times for 30 min each time in 2 x SSC-0.5% SDS at 66 C and twice for 30 min each time in 0.1 x SSC-0.1% SDS at 66 C, and exposed at -80 C for 3 days. The sizes of the transcripts and loading efficiencies were determined by comparison with ribosomal RNA bands.

Hormone determinations
Serum T was measured by RIA as previously described (29, 37, 38). Intra- and interassay coefficients of variation were 8.5% and 11.2%, respectively, and the detection limit was 0.67 nmol/liter. Rat FSH was determined by RIA as outlined previously (37, 38). The detection limit was 0.78 µg/liter, and the intraassay coefficient of variation was 7.3%. FSH-dependent inhibin concentrations had been determined previously (29, 30). The detection limit was 1.2 µg/liter, and intraassay variation was 2.8%.

Statistical evaluation
Data from VEH and ANT groups were compared by two-sample t test. P < 0.05 was considered significant. Data are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of CREM expression in monkey testis (Figs. 1Go and 2Go)
CREM expression was seen in round spermatids, and expression was maximal during stages V–VII and declined thereafter. In stages VIII and IX, some round spermatids were CREM positive, whereas others were negative, and overall staining intensity declined. Spermatid elongation is initiated in these stages. A weak but specific signal for CREM was present in nuclei and cytoplasm of pachytene spermatocytes during stages VIII–XI and during meiotic divisions (stage XII).



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  		Figure 1. Immunocytochemical localization of CREM expression in monkey testis. Neufuchsin was used for the detection of CREM (pink and red), and hematoxylin was used as counterstain. a–d, VEH treatment; e–h, ANT treatment. a, Low power micrograph showing stage-specific CREM expression. b, CREM expression is present in spermatids (short arrow) and in pachytene spermatocytes (asterisk). The long arrow points to spermatocytes in meiotic division. Signal intensity in spermatids is stage dependent. c, Strong CREM expression was found in round spermatids in a stage VI tubule (short arrow), whereas in spermatids in a stage VIII tubule, expression was reduced, and it was nearly absent in spermatids (asterisk) of a stage IX tubule. Pachytene spermatocytes express CREM in stage VIII (long arrow). d, Preincubation of primary antibody with human recombinant CREM protein abolished the CREM signal. e, Twenty-five days of ANT treatment. Stage-related organization of the germinal epithelium is still present. Expression of CREM is retained (short arrow). Leydig cells are involuted (asterisk), and the numbers of early spermatocytes are markedly reduced (long arrows). f, Severe involution after 25 days of ANT resulted in severe spermatogenic involution in one animal. Although some elongated spermatids are retained (long arrows) the number of spermatocytes and round spermatids is pronouncedly lowered. Note that the remaining round spermatids express CREM (short arrows). g, Low power micrograph after 56 days of ANT treatment. Testicular involution is extreme, but round spermatids exhibiting CREM expression are still present (short arrows). h, Fifty-six days of ANT. Mainly spermatogonia and Sertoli cells are present. Spermatocytes are rare, the number of round spermatids is low, and elongated spermatids are absent. CREM expression is still present in some round spermatid nuclei (short arrows).

 


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  		Figure 2. Image analysis of CREM expression in monkey testis. Data represent the mean ± SEM of three animals treated either with VEH (open circles) or ANT (closed circles) for 25 days. As not all stages could be identified unequivocally, some stages were combined. The upper panel represents the stage-dependent organization of the macaque seminiferous epithelium, and CREM-positive germ cells are denoted by a shaded bar. The middle panel refers to data obtained for spermatids, and the bottom panel refers to data obtained for spermatocytes. Data are expressed in arbitrary units based on the equipment-inherent gray scale, and differences are not significant (P > 0.05).

 
Localization of CREM expression in rat testis (Figs. 3Go and 4)
CREM expression was seen in round spermatids, and the strongest signals were found during stages IV–VII. Signal intensity declined during stages VIII–X coinciding with the start of spermatid nuclear elongation, and CREM expression was absent in spermatids during stages XI–XIV. A weak CREM signal was observed in the nuclei and cytoplasm of pachytene spermatocytes during stages IX–XIII and during meiotic division (stage XIV).



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  		Figure 3. Immunocytochemical localization of CREM expression in rat testis. Neufuchsin was used for detection of CREM (pink and red), and hematoxylin was used as a counterstain. a–d, VEH treatment; e–g, ANT treatment; h, hypophysectomy. a, Low power micrograph showing stage-specific CREM expression. b, CREM expression in spermatid nuclei (short arrows) and signal intensity are related to spermatogenic stage. The asterisk denotes a cluster of Leydig cells. c, CREM expression in dividing late spermatocytes (long arrow) and is intense in round spermatids at particular spermatogenic stages (short arrow). Pachytene spermatocytes also express CREM. d, Preincubation of primary antibody with human recombinant CREM protein abolished the CREM signal. e, ANT for 14 days. Stage-related organization of the germinal epithelium and CREM expression are retained (short arrow). Leydig cells are involuted (asterisk), and degenerating germ cells appear in stage VII tubules (long arrows). Tubular size is reduced (same magnification as in b and c). f, Forty-two days of ANT. Severe involution of the germinal epithelium. Only round spermatids that express CREM are retained (short arrows). The long arrow denotes late pachytene spermatocytes during meiotic division. Weak CREM expression is evident. g, High power micrograph after 42 days of ANT. The asterisk denotes B-type spermatogonia, indicating that the round spermatids in these tubules are in stages IV–VI. CREM expression is intense (short arrow). h, Fourteen days after hypophysectomy. Seminiferous epithelial involution is obvious, and spermatogenesis is interrupted. This tubule contains some preleptotene spermatocytes, indicating that the corresponding round spermatids could belong to a stage VII or VIII tubule. CREM expression is evident (short arrow) in some spermatids.

 
CREM expression in ANT-suppressed monkeys (Figs. 1Go and 2)
After 25 days of ANT, round spermatid numbers were reduced by about 10% (30), and spermatogenic stages could still be recognized. CREM expression was unchanged compared with that in the VEH group. After 56 days of ANT treatment, spermatogenesis was severely disrupted. Round spermatids were seen in some tubules only. CREM signal intensity in these cells was 19,808 ± 2,714 (n = 217 cells). Due to the disorganization of the seminiferous epithelium and the limited size of the testicular biopsy, stage-related analysis was not possible.

CREM expression in ANT-suppressed and hypophysectomized rats (Figs. 3Go and 4Go)
After 14 days of ANT, numbers of elongating and elongated spermatids were visibly reduced. The CREM staining pattern was similar to that in the VEH group. After 42 days of ANT, the most advanced germ cells present were round spermatids. These cells were consistently positive for CREM, and signal intensity was 26,854 ± 1,461 (n = 268 cells). Because of the pronounced loss of spermatids, stage-related analysis could not be performed. However, based on the presence of B-type spermatogonia and preleptotene spermatocytes, round spermatids probably attained stages V–VIII. Pachytene and dividing (stage XIV) spermatocytes exhibited a clear CREM signal. The CREM signal in round spermatids was maintained 14 days after hypophysectomy (28,351 ± 2,053; n = 138 cells) and during FSH supplementation (28,958 ± 1,784; n = 115 cells).



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  		Figure 4. Image analysis of CREM expression in rat testis. Data represent the mean ± SEM of three animals treated with either VEH (open circles) or ANT (closed circles) for 14 days. As not all stages could be identified unequivocally, some stages were combined. The upper panel represents the stage-dependent organization of the rat seminiferous epithelium, and CREM-positive germ cells are denoted by the shaded bar. The middle panel refers to the data obtained for spermatids, and the bottom panel refers to data for spermatocytes. Data are expressed in arbitrary units based on the equipment-inherent gray scale, and differences are not significant (P > 0.05).

 
Western blot analysis (Figs. 5Go and 6Go)
A major band of approximately 35 kDa was present in monkey testis tissue. This band was preserved in ANT-exposed testis. An additional smaller band was observed in one control animal (Fig. 5Go, lane 1). In the rat testis, several bands were present on Western blots, and expression of these bands was retained during ANT treatment.



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  		Figure 5. Western blot analysis of CREM in nonhuman primate testis. Four hundred micrograms of testicular protein were loaded. Animals were treated with VEH or ANT for 25 days. Lane 4 represents polyhistidine-tagged human CREM protein (3 µg), and lane 5 contains molecular mass marker. A major band is present at 35 kDa in both treatment groups.

 


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  		Figure 6. Western blot analysis of CREM in rat testis. Four hundred micrograms of testicular protein were loaded. Animals were treated with VEH or ANT for 14 days. Lane 4 represents polyhistidine-tagged human CREM protein (3 µg), and lanes 5 and 11 contain molecular mass marker. CREM expression is maintained despite ANT exposure. Lane 9, Rat testis extract probed with antibody that had been preincubated with human CREM; lane 10, polyhistidine-tagged human CREM protein (3 µg) was loaded and probed with antibody that had been preincubated with human CREM.

 
Northern blot analysis (Figs. 7Go and 8Go)
Several CREM transcripts around 2 kb were present in monkey testis and were retained after ANT exposure for 25 days. In the rat testis, CREM transcript signals were similar after VEH and ANT treatments.



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  		Figure 7. Northern blot analysis of CREM in monkey testis. Twenty micrograms of total testis RNA were loaded. Animals were treated with VEH or ANT for 25 days. Note that RNA was degraded in one control sample.

 


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  		Figure 8. Northern blot analysis of CREM in rat testis. Twenty micrograms of total testis RNA were loaded. Animals were treated with VEH or ANT for 14 days. The bottom panel represents ribosomal RNA bands to indicate loading efficiency.

 
Antibody specificity for CREM
Omission of primary antibody or preincubation with recombinant CREM (Figs. 1dGo and 2dGo) abolished the cytoplasmic and nuclear CREM signals. To clarify whether the antibody recognizes related proteins, i.e. CREB, CREM expression was studied in CREM-deficient mice lacking the DNA-binding domain and the nuclear targeting signal (18) thereby preventing translocation into the nucleus. Nuclear staining for CREM was absent, whereas weak but distinct cytoplasmic CREM expression was observed (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This work describes the expression and localization of CREM in rat and monkey testes and investigates whether testicular CREM expression is dependent on the pituitary hormones LH and FSH. Nuclear CREM signals were strongest in spermatids in spermatogenic stages IV–VII in the rat and V–VII in the monkey. Although the stage classification of spermatogenesis is different (14 stages in rats and 12 stages in monkeys), the pattern of CREM expression was similar when related to spermatid development. CREM expression attained peak levels in those stages associated with the flattening of the acrosomal vesicle and formation of the acrosome (39), and disappeared at the initiation of nuclear elongation. It is noteworthy that high expression levels of CREM coincided with the appearance of a testis-specific form of an actin-capping protein that contains a putative CRE element (40). These proteins control cytoskeletal activity and cell shape. In mice lacking a functional CREM gene, spermatogenesis proceeded to the development of early round spermatids (18, 19) and CREM expression can be altered in patients with spermatid maturation defects (26). Collectively, these findings strongly indicate that CREM functions as a key regulator of spermatid development and maturation in the mammalian testis.

The CREM antiserum used in the present investigation recognizes CREM isoforms with similar affinity, but shows only weak cross-reactivity with CREB (21). The latter observation is confirmed by the present work, as no nuclear signal was obtained with CREM antiserum in CREM-deficient mice (18). Unlike Delmas et al. (21), we detected a specific immunocytochemical CREM signal in late spermatocytes. These different findings might be related to the sensitivity of the immunocytochemical techniques, as the (extra)avidin/biotin-enzyme system is highly sensitive (41). Transcripts of CREM{tau} activator were present in pachytene spermatocytes (20), whereas CREM repressors were absent from pachytene spermatocytes and round spermatids (20, 21). As CREM signal vanished after preincubation of primary antibody with CREM, the spermatocyte signal is considered to represent a CREM antigen. It is puzzling, however, that CREM in these germ cells was also localized in the cytoplasm, as CREM is a nuclear protein. A cytoplasmic form of the closely related CREB molecule has been described in rat testis that lacks the DNA-binding domain and the nuclear import signal, but its functional role is yet unknown (42). At present, it can only be assumed that the observed CREM signal might represent cytoplasmic CREM, and further characterization of this signal is warranted.

ANT administration was used because these compounds suppress LH and FSH secretion in all mammalian species studied to date (2). For combined analysis by immunocytochemistry and Western and Northern blots in rat and monkey, a design was chosen to achieve deficient gonadotropin secretion but to preserve the spermatogenic cycle (37, 38, 43, 44, 45). FSH-dependent formation of B-type spermatogonia (43, 46, 47) was reduced to 10% of the control value (30), providing compelling biological evidence that the action of FSH had been eliminated. Despite severe gonadotropin deficiency, stage- and germ cell-specific CREM expression was retained, and CREM signal intensity persisted even after prolonged (6–8 weeks) gonadotropin suppression. We cannot exclude the possibility that CREM proteins detected in this study were not phosphorylated and that FSH is necessary for phosphorylation and activation of CREM activator proteins. Round spermatid CREM expression was also quantitatively retained 2 weeks posthypophysectomy, and FSH did not alter CREM signal intensity. Altogether, it appears that testicular expression of CREM in haploid germ cells is not dependent of gonadotropins but, rather, is related to the developmental stage of spermatid maturation.

Our observations are at variance with those by Foulkes and colleagues (22), who reported a marked reduction or absence of CREM{tau} expression either 1–2 weeks posthypophysectomy in rat or after 10-week exposure to short photoperiod in hamsters, and restoration of expression by FSH within 3 h in both species. As testicular histology was not described in the experiments by Foulkes et al. (22), it remains unknown whether hypophysectomy lowered germ cell numbers to such an extent that Northern blot analysis of total testicular RNA was not sufficient to obtain positive signals. Spermatocytes and some early spermatids were still present in hamsters maintained for 10 weeks under reduced day length (Desjardin, C., et al., 1971, quoted from Ref. 22). As testis size had dropped by 85–90% (22), the number of remaining spermatids can indeed be presumed to have been very low. However, the reason(s) for the discrepant findings obtained in the present study and in earlier investigations (22) remain unclear. Nonetheless, results from other investigations lend support to the view that testicular CREM expression is not under gonadotropin control. We identified infertile men in whom testicular CREM expression was absent, although serum FSH concentrations were normal or elevated (26). Men bearing an inactivating mutation of the FSH receptor (3) and mice lacking the FSH ß-subunit (4) or the FSH receptor (5) are fertile. Indeed, CREM expression was unaltered in the latter investigation. It should be noted that FSH, in conjunction with LH/T, acts to maintain quantitatively normal spermatogenesis rather than specifically on a particular gene or germ cell, and that the role of FSH alone in male gametogenesis is still controversial (3, 4, 5, 6).


   Acknowledgments
 
The authors are grateful to Dr. E. Nieschlag, Institute of Reproductive Medicine (Munster, Germany), for continued support. We are thankful to Dr. G. Schütz, Molecular Biology of Cell I, German Cancer Research Center (Heidelberg, Germany), for critical comments. The GnRH antagonist cetrorelix was a generous gift from Dr. Th. Reissmann, ASTA Medica (Frankfurt, Germany). We are indebted to S. Nieschlag, M.A., for language editing. The expert technical assistance of Agnes Rösner, Reinhild Sandowe, Guenter Stelke, and Martin Heuermann is gratefully acknowledged. We acknowledge the support of Dr. G. Clemen (Institute of Special Zoology and Comparative Embryology of the University of Munster).


   Footnotes
 
1 This work is part of the doctoral thesis by R.B. at the Fachbereich Biology of the University of Munster and was supported by the Deutsche Forschungsgmeinschaft Confocal Research Group, The Male Gamete: Production, Maturation, Function (Ni-130/15–2). Back

Received August 31, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sharpe RM 1994 Regulation of spermatogenesis. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, pp 1363–1434
  2. Weinbauer GF, Nieschlag 1996 The Leydig cell as a target for male contraception. In: Payne AH, Hardy MP, Russell LD (eds) The Leydig Cell. Cache River Press, Clearwater, pp 629–662
  3. Tapanainen JS, Aittomäki K, Min J, Vaskivuo T, Huhtaniemi IT 1997 Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nat Genet 15:205–206[CrossRef][Medline]
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