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Cyclic Adenosine 3',5'-Monophosphate-Responsive Element Modulator Gene Expression in Germ Cells of Normo- and Oligoazoospermic Men

Endocrine (A.P., F.C., M.S.) and Andrology Unit (C.K., S.G., G.F.), Department of Clinical Physiopathology, University of Florence School of Medicine, 50139 Florence; and the Department of Internal Medicine, Andrology Section, University of L’Aquila (S.F.), 67100 L’Aquila, Italy

Address all correspondence and requests for reprints to: Alessandro Peri, M.D., Ph.D., Endocrine Unit, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. E-mail: a.peri{at}dfc.unifi.it

	Abstract

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In about one third of infertile men the cause of impaired spermatogenesis is not known. Spermatogenesis appears to be mediated at least in part by the pituitary gonadotropins, which activate the cAMP-dependent signaling pathway. The end point of this pathway is the activation of nuclear transcription factors, such as cAMP-responsive element-binding protein and cAMP-responsive element modulator (CREM). These factors, upon binding to gene sequences identified as cAMP response elements, modulate the expression of germ cell-specific genes that, in turn, promote the completion of spermatogenesis. The expressions of the cAMP-responsive element-binding protein and CREM genes create different isoforms, which can be divided into two groups: activators or repressors of gene regulation. Only CREM repressors are expressed in premeiotic germ cells in mice, whereas a switch to the expression of the CREM activator is observed from postmeiotic germ cells onward. Completion of germ cell maturation appears to be dependent on this phenomenon. Recently, mice lacking CREM gene expression have been generated. These animals were infertile and presented a developmental arrest of germ cell maturation at the stage of early spermatid. In this report we demonstrate that CREM gene expression also occurs in human germ cells. In particular, we determined by RT-PCR that a switch from the expression of CREM repressors to CREM activators is present in postmeiotic germ cells in normospermic men. Conversely, in oligoazoospermic patients only the expression of CREM repressors was detected. These data were confirmed by in situ hybridization studies in which transcripts for CREM activators were detected in postmeiotic germ cells in testis specimens showing conserved spermatogenesis, but not in specimens showing maturation arrest at the spermatid stage. Thus, our results indicate that the lack of a switch in the expression of CREM gene isoforms may be related to impaired spermatogenesis in humans.

	Introduction

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INFERTILITY affects about 15% of couples during reproductive age, and this phenomenon appears to be distributed about equally between the sexes (1). It is noteworthy that in about one third of the cases of infertility due to the male partner the cause of impaired spermatogenesis is not known; therefore, these patients are reported as affected by idiopathic infertility (2). Spermatogenesis, the developmental process by which immature germ cells differentiate into mature fertilizing spermatozoa is dependent on the activation of cAMP-dependent signaling pathways in the testis, mediated by the pituitary gonadotropic hormones FSH and LH (3, 4). A final step in the cAMP cascade is the activation of nuclear transcription factors cAMP-responsive element-binding protein (CREB) and cAMP-responsive element modulator (CREM). These factors bind to nuclear cAMP response elements (CRE), thus modulating the expression of germ cell-specific genes such as protamine-1 and -2, transition protein-1 and -2, and calspermin (5, 6, 7, 8, 9, 10). These proteins are required for structuring of mature spermatozoon (11). CREB and CREM gene expressions create different isoforms by mechanisms of alternative exon splicing, alternative promoter usage, and autoregulation of promoters (7, 12). The different isoforms can be divided into two groups: activators or repressors of gene expression. It has been shown that a switch in CREM expression occurs during spermatogenesis. Indeed, only CREM repressors are expressed at low amounts in premeiotic germ cells, whereas transcripts for the CREM activator are detected starting from pachytene spermatocytes (13), and the related protein is present in high amounts in spermatids. This event appears to be responsible for completion of germ cell maturation. Recently, this hypothesis has been confirmed by generating CREM mutant mice through homologous recombination (14, 15). The analysis of the seminiferous epithelium in mutant mice revealed development arrest at the round spermatid stage.

Spermatogenic arrest is also a feature of several cases of infertility in human males (11). Thus, in the present study we sought to determine whether the CREM gene is expressed in human germ cells and whether a different expression pattern can be envisaged in normospermic and oligoazoospermic subjects.

	Subjects and Methods

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Patients

Seminal fluid obtained by masturbation from six oligoazoospermic patients (0–1.7 x 10⁶ sperm/mL seminal fluid) was used for germ cell separation and polyadenylated [poly(A)⁺] ribonucleic acid (RNA) extraction for subsequent RT-PCR studies. Three normospermic volunteers (50–80 x 10⁶ sperm/mL seminal fluid) served as normal controls. The clinical features of the patients are presented in Table 1. Blood was collected from a volunteer for subsequent poly(A)⁺ RNA extraction from leukocytes. For in situ hybridization experiments, testicular biopsy specimens (n = 5) were used. Two subjects had a testicular histology of conserved spermatogenesis (obstructive azoospermia), and three patients had a histology of round spermatid maturation arrest. Informed consent was obtained from the patients.

Ejaculates were obtained by masturbation after 3–4 days of sexual abstinence and were left to stand for 30 min at laboratory temperature to allow liquefaction. Whole samples were than loaded on top of discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient column consisting, from the bottom, of 2 mL 40% Percoll, 1 mL 50% Percoll, 1 mL 75% Percoll, and 1 mL 90% Percoll in HTF medium (Irvine Scientific, Santa Ana, CA). After centrifugation at 330 x g for 20 min, individual fractions were resuspended in 0.5 mL HTF medium and counted. For each fraction an aliquot was spread on prestained slides (Testsimplets, Boehringer Mannheim, Italia, Monza, Italy) for characterization of cells.

RT-PCR

RT-PCR was performed using poly(A)⁺ RNA extracted from germ cells. Germ cells were fractionated on Percoll gradient before RNA extraction, as previously described. Poly(A)⁺ RNA was extracted using a commercially available kit [messenger RNA (mRNA) Capture Kit, Boehringer Mannheim]. This kit allows the capture of poly(A)⁺ RNA directly onto test tubes for PCR. Poly(A)⁺ RNA was extracted from 120 x 10³ ejaculated germ cells/test tube and from 10 x 10⁶ leukocytes/tube. To perform RT-PCR, the Titan RT-PCR System (Boehringer Mannheim) was used according to the manufacturer’s instructions. The CREM-specific primers were: CREM-L (sense primer), 5'-GGAAACAGTTGAATCCCAGC-3'; and CREM-R (antisense primer), 5'-AGGCACATCAGAGGACAGTT-3'. These primers span sequences of exons B and D, respectively, and were designed to create two different signals, corresponding to the inhibitory isoforms , ß, and and the activating isoforms , , and 1 of the CREM gene, respectively. As exon C, which is located between exons B and D, is expressed only in the activating isoforms, these are readily separated by gel electrophoresis from the inhibitory isoforms by means of the different RT-PCR product length (390 vs. 243 bp). The quality of the poly(A)⁺ RNAs used was assessed by performing additional RT-PCR using primers specific for the glyceraldehyde-3-phosphate dehydrogenase gene (16).

RT-PCR products were subjected to Southern blotting and subsequent hybridization using a CREM-specific oligonucleotide as the probe (CREM-P). CREM-P spans a sequence internal to exon B, thus allowing the detection of either CREM activators or repressors. The sequence of the probe was 5'-GCAGAATCAGAAGGTGTAAT-3'. The hybridized DNAs were detected using an immunochemiluminescent method (Boehringer Mannheim), as previously described (13).

In situ hybridization

These experiments were performed as previously described (17). Briefly, fresh-frozen testicular biopsy specimens were placed on ribonuclease-free slides and kept at -80 C until the day of the experiment. Two different 48-mers (CREM-AP and CREM-SP), synthesized by Med Probe (Oslo, Norway), were used as the probes. The probes were 3'-end labeled with digoxigenin-11-deoxy-UTP using the Oligonucleotide Tailing Kit (Boehringer Mannheim). The hybridized mRNAs were detected using an immunocolorimetric method (Boehringer Mannheim), previously described (17). The slides were examined using a Nikon Microphot FX microscope (Nikon, Melville, NY).

	Results

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RT-PCR studies

RT-PCR experiments were performed using poly(A)⁺ RNA isolated from germ cells at different stages of maturation to assess the presence of transcripts for either the activating or the inhibitory CREM isoforms. Cells were separated in different fractions as described in Materials and Methods. Ejaculated germ cells were obtained from volunteers with normal semen parameters (n = 3) as well as from subjects presenting azoospermia or severe idiopathic oligospermia (n = 6). The clinical features of the patients are presented in Table 1. A typical example of an experiment performed using germ cells from a normal volunteer is shown in Fig. 1A. A signal of 243 bp, corresponding to the inhibitory isoforms , ß, and of CREM, was obtained from RNA extracted from the Percoll fractions containing mostly germ cells at early stages of differentiation (40–50%). No 243-bp signal was present in the 75% fraction of the Percoll gradient, which contains predominantly spermatids, whereas a signal (390 bp) corresponding to the activating CREM isoforms , , and 1 was detected in this fraction. Faint signals corresponding to either the 243- or 390-bp transcripts were detected in the 90% fraction (mostly mature germ cells). These results are in keeping with the switch in the expression of CREM gene isoforms observed in mouse spermatogenesis (10). Conversely, RT-PCR analysis for the expression of CREM isoforms in germ cells from oligoazoospermic patients revealed only the presence of a signal corresponding to the inhibitory isoforms (243 bp), as shown in Fig. 1B, in which the results of a typical experiment are represented. Only in the case of an oligospermic patient was the simultaneous presence of the 243- and 390-bp signals observed in the 75% Percoll gradient fraction, whereas, according to the other cases examined, only the 243-bp signal was detected in the other Percoll fractions (Fig. 2). This may have been due to the presence of conserved spermatogenesis in some seminiferous tubules of this oligospermic patient. The fact that the pair of primers used in our study can simultaneously create two different products provides a way to quantitatively estimate the results based on the intensities of the two signals, thus circumventing the necessity of assessing RNA quality. However, the homogeneous good quality of the poly(A)⁺ RNAs used in our study was confirmed by subjecting all RNAs to RT-PCR for analysis of the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase; the results of this were virtually identical in all of the samples examined (not shown). In addition, to exclude that our results could be invalidated by the presence of leukocytes in the seminal fluid collected for RNA extraction, RT-PCR for the detection of CREM-specific transcripts was performed using poly(A)⁺ RNA extracted from leukocytes collected from blood sampling. Under our experimental conditions, by using poly(A)⁺ RNA extracted from a number of leukocytes, exceeding by 80-fold the number of germ cells from which poly(A)⁺ RNA was usually extracted (10 x 10⁶ vs. 120 x 10³), only a very faint 243-bp signal, corresponding to the inhibitory CREM isoforms, was observed (not shown). Therefore, we showed that the presence of leukocytes as a contaminant in seminal fluid could not interfere with the results shown.

View larger version (20K): [in this window] [in a new window]   Figure 1. RT-PCR on ejaculated germ cells for CREM gene expression assessment. A, Ethidium bromide-stained agarose gel, showing CREM gene expression in germ cells of a normospermic man; B, CREM gene expression in germ cells of an azoospermic (40*) and an oligospermic patient (other lanes); C, chemilumigram showing the results of the same experiment reported in A, after hybridization with CREM-P. ST, DNA molecular weight marker VI (Boehringer Mannheim); N, no RNA control reaction; 40–90, percentage of the Percoll gradient.

View larger version (44K): [in this window] [in a new window]   Figure 2. Ethidium bromide-stained agarose gel, showing RT-PCR products for CREM gene expression on ejaculated germ cells of an oligospermic patient. ST, DNA molecular weight marker VI; N, no RNA control reaction; 50–90, percentage of the Percoll gradient.

View larger version (43K): [in this window] [in a new window]   Figure 3. Chemilumigram showing the hybridization pattern for CREM-P in an oligospermic patient. St, DNA molecular weight marker VI (Boehringer Mannheim); N, no RNA control reaction; 40–90, percentage of the Percoll gradient.

RT-PCR studies allowed us to evaluate the presence of specific transcripts in poly(A)⁺ RNAs extracted from cells separated on a Percoll gradient. Germ cells fractionated in this way are representative of specific germ cells that are predominant in each Percoll fraction, but cannot be considered the expression of a completely homogeneous cell population. Therefore, to further investigate the presence of CREM-specific transcripts in germ cells and, in particular, to detect and locate the presence of specific activating isoforms, in situ hybridization studies were performed on testicular biopsies (Fig. 4). Testis specimens were obtained from either patients with a histological diagnosis of normal spermatogenesis (obstructive azoospermia; n = 2; Fig. 4, A, C, and D) or maturation arrest at the level of the round spermatid (n = 3; Fig. 4, B, E, and F). In Fig. 4, A and B, hematoxylin-eosin-stained specimens for histological assessment are shown. In Fig. 4, C and E, photomicrographs after hybridization with a 48-mer probe specific for CREM-activating isoforms (CREM-AP) are shown. In Fig. 4C, evident staining is present in the cytoplasm of postmeiotic germ cells. The nuclei of the cells were counterstained with methyl green. In the tubule represented in Fig. 4E (round spermatid arrest), no CREM-specific staining is evident in germ cells. No staining was detectable in sections hybridized to the sense probe (CREM-SP) as negative controls (Fig. 4, D and F). Other negative controls were performed by omitting the antisense probe (not shown). This hybridization pattern was virtually identical in the other cases examined.

View larger version (169K): [in this window] [in a new window]   Figure 4. In situ hybridization for CREM gene expression detection in human testis. A, C, and D, Obstructive azoospermia; B, E, and F, spermatid arrest. A and B, Hematoxylin-eosin-stained sections; C and E, antisense CREM probe-hybridized sections; D and F, sense CREM probe-hybridized sections. The arrows indicate postmeiotic germ cells. Magnification: A and B, x50; C and D, x200; E and F, x100.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Our results were substantiated by in situ hybridization experiments, performed on testicular biopsies. This approach allowed us to target the expression of CREM-activating isoforms in specific germ cells and therefore appears to be the elective technique to determine the localization of a transcript in a incompletely homogeneous cell population. The probe we used, spanning a sequence internal to exon C, unequivocally hybridized to mRNA sequences common to CREM , , and 1 in postmeiotic germ cells of patients with obstructive azoospermia, with conserved spermatogenesis. The specificity of the staining was determined by the absence of positivity in adjacent sections hybridized to the sense probe. Conversely, in patients with a histological assessment of maturation arrest at the level of the round spermatid, germ cells definitively did not show any positivity for CREM activator transcripts. These results confirm the RT-PCR data, indicating that a switch to expression of the activating isoforms of CREM gene is coupled to the completion of spermatogenesis in humans. Despite the superior reliability of in situ hybridization techniques over RT-PCR for targeting a germ cell maturation arrest as the result of inappropriate gene expression, the analysis of CREM transcripts in poly(A)⁺ RNAs from ejaculated germ cells could, as was shown to be the case in our hands, provide preliminary information and could be performed as a first screening diagnostic procedure or in those laboratories in which testicular biopsies are not routinely performed.

Recently, our results were supported by the work of another group (18), which reported the absence of or severely impaired immunostaining for CREM protein in men with spermatid maturation arrest compared to normospermic volunteers. In addition, another recent study showed the presence of a 32-kDa putative actin-capping protein, possibly related to the development of the final shape of mature sperm heads (19), in mouse testis. The expression of this protein appeared to be under the control of the cAMP cascade, and the immunohistochemical positivity was uniquely located in the cytoplasm of round spermatids.

In conclusion, in the present report we have shown for the first time, although preliminarily, that the lack of transcripts for CREM-activating isoforms in postmeiotic germ cells can be linked to impaired spermatogenesis in humans and could be addressed as one of the possibly multiple causes of infertility.

	Acknowledgments

 
The authors thank Prof. Gabriella Barbara Vannelli, Human Anatomy Department, University of Florence, for her expert assistance with the microphotography work.

Received March 5, 1998.

Revised June 2, 1998.

Accepted June 15, 1998.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Peri, A. Articles by Serio, M. Search for Related Content PubMed PubMed Citation Articles by Peri, A. Articles by Serio, M.