Immunohistochemical measurement and expression of Mcl-1 in infertile testes

Abstract

The diagnosis of azoospermia represents a major challenge to andrologists as this condition may occur despite normal spermatogenesis and genital tracts. Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins involved in regulation of apoptosis in various cell types. This study aimed to investigate the immunohistochemical expression of Mcl-1 in testicular biopsies of subjects with azoospermia. Eighty-six cases with azoospermia were obtained from 509 infertile patients admitted to the Andrology Unit of the Zagazig University Hospitals from January 2010 to December 2011. Biopsies were diagnosed and classified using H&E-stained slide sections. The specimens were subjected to immunohistochemical staining for Mcl-1 and examined through light microscopy. Forty-five cases of maturation arrest (25 at spermatids and 20 at the spermatocytes), 31 cases of hypospermatogenesis (20 moderate and 11 severe), 5 cases of Sertoli cell-only syndrome, 2 cases of basement membrane hyalinization, and 1 case of tubular and peritubular sclerosis were observed. Normal spermatogenesis was detected in 2 cases. A strong positive immunoreaction in Leydig cells was observed among all investigated specimens. A moderate reaction was detected in spermatocytes and spermatozoa in cases of normal spermatogenesis and hypospermatogenesis, but a negative reaction was detected in cases of maturation arrest and germ cell aplasia. Apoptosis was found to be associated with decreased rate of spermatogenesis. High apoptosis rates may result in azoospermia, which can occur despite normal spermatogenesis and absence of duct obstruction.

Keywords

spermatogenesis / testis / Mcl-1 expression / immunohistochemical study / infertility

Introduction

A fine balance between cell proliferation and cell loss is necessary for normal tissue homeostasis. Apoptosis is a strictly regulated process important for removal of aged, damaged, and unnecessary cells during normal animal development; apoptosis deregulation is associated with the pathogenesis of a wide range of diseases [ 1]. Apoptosis is morphologically characterized by nuclear fragmentation, formation of membrane-encased apoptotic bodies containing organelles, and cell shrinkage [ 2].

Spermatogenesis is a complex biological process of cellular transformation, in which spermatogonia (stem cells) divide and differentiate into mature spermatozoa. The relative inefficiency of human spermatogenesis has been well documented; the human testis produces low numbers of sperm cells per unit weight of testis compared with various animal species [ 3], but factors affecting this inefficiency remain unknown. In animal studies, germ cell deletion during normal spermatogenesis results in losses of up to 75% of the potential mature sperm cells in the adult testis [ 4]. Apoptosis is probably the major mechanism for such cell loss. Despite the putative implications of apoptosis for improved fertility control and clinical management of infertility in men, the mechanisms of germ cell death remain poorly understood [ 5].

Apoptosis is the underlying mechanism of germ cell death during normal spermatogenesis and can be triggered by a wide variety of regulatory factors, including the Bcl-2 family of proteins. Myeloid cell leukemia-1 (Mcl-1) is a member of this family and presumed to be an anti-apoptotic gene [ 6]. Induced deletion of Mcl-1 in mice results in ablation of bone marrow [ 7]. Thus, Mcl-1 could be a critical and specific regulator to ensure the homeostasis of early hematopoietic progenitors. This study aimed to investigate Mcl-1 expression in infertile testes of subjects with azoospermia.

Patients and methods

Patients and testicular biopsies

A total of 509 patients admitted to the Andrology Unit, Faculty of Medicine, Zagazig University Hospitals between January 2010 and December 2011 were recruited. Complete clinical data, including age, fertility history, clinical examination, hormonal profile, and semen analysis results, of each patient were recorded. Eight-six patients who suffered from azoospermia were selected for the study, after their written consents were obtained. The age of these patients ranged from 22 to 35 years, and the size of their testes was within the normal range. These patients manifested primary infertility but presented normal hormonal profiles without apparent obstruction of the vas deference or tubules. The selected patients had no history of diseases or medications that may affect spermatogenesis. Testicular biopsy was performed.

Histological preparation

Tissue was fixed in Bouin’s solution for 4 h and in neutral buffered formalin for 12 h. The sections were processed in alcohol and xylene and then embedded in paraffin. The histological features of all cases were studied in slides stained with hematoxylin-eosin (H&E). Histological diagnosis was performed according to the criteria reported in the literature [ 8]. The findings were histopathologically classified as normal spermatogenesis, hypospermatogenesis (mild, moderate, or severe), maturation arrest, germinal cell aplasia (Sertoli cell-only syndrome), hyalinization (cellular elements disappeared, leaving thickened seminiferous tubule walls), or sclerosis [ 9, 10].

Immunohistochemistry

Immunohistochemistry

Immunohistochemical staining was performed as described by Boenisch et al. [ 11]. Briefly, (1) the tissue sections were deparaffinized by washing twice in xylene for 5 min each. (2) The sections were rehydrated in graded ethanol and (3) incubated in 3% (v/v) H₂O₂ in methanol for 5 min to block endogenous peroxidase. (4) The sections were then washed in phosphate-buffered saline (PBS) for 5 min. (5) For antigen retrieval, the sections were heated in a microwave for 10 min in 10 mmol/L citric acid buffer (pH 6.0) and rinsed with lukewarm tap water. (6) The sections were placed in Tris-buffered saline (TBS, pH 7.8) for 5 min and blocked in Tris-NaCl-KCl (TNK) buffer (100 mmol/l Tris, pH 7.6–7.8, 550 mmol/L NaCl, 10 mmol/L KCl) containing 2% (w/v) bovine serum albumin (BSA), 0.1% Triton X-100, and 1% normal goat or donkey serum. (7) For application of primary antibody, the rabbit anti-human Mcl-1 polyclonal antibody (Santa Cruz Biotechnology, CA, USA; 1:10 and/or 1:20 dilutions of 100 μg/ml stock in TNK buffer) was added. The sections were incubated overnight in a humidified chamber placed at 4 °C and then (8) washed once with PBS. (9) For application of biotinylated secondary antibody, the sections were incubated for 1 h at room temperature in a humidified chamber with the biotinylated goat anti-rabbit antibody in TNK buffer (1:500; Vector Laboratories, Burlingame, CA, USA). (10) The sections were then washed with PBS. (11) For application of the streptavidin-biotin complex, the sections were incubated for 30 min at room temperature with streptavidin-horseradish peroxidase in TNK buffer (1:20). (12) For color development, the sections were incubated in a development solution containing 0.06% DAB and 0.1% (v/v) H₂O₂ in TNK buffer (without serum, BSA, and Triton X-100) for 10 min. (13) Finally, the sections were counterstained with hematoxylin and then mounted and microscopically examined. For each batch of Mcl-1 staining evaluation, positive-control sections from a case with breast cancer and negative-control sections from testicular specimens without the primary antibody (Mcl-1) were included.

Scoring and percentages

The two parameters evaluated are the intensity of nuclear staining and the extent/percentage of positive cells. The fields for calculating the percentage of immunoreactive nuclei contained no artifacts, wrinkling, or folding. As immunoreactivity may not be uniform in the cytoplasm or membrane for any given case, the most frequently observed pattern was graded. The intensity of cytoplasmic/membranous staining was graded as no staining (0), weak (1+), moderate (2+), or strong (3+). Extent was semiquantitatively estimated from 0% to 100%. Percentages were determined by counting at least 500 cells (with the calibrating eye piece) and establishing the ratio of immunoreactive nuclei to the total number of cells multiplied by 100. These percentages were rounded off to the nearest 10%. Scoring criteria were presented as follows: less than 10% positive cells= 0; 10%–30% positive cells= 1; 31%–60% positive cells= 2; and more than 60% positive cells= 3. Statistical analysis was based on the data distribution for a continuum from 1%–100% reactive cells (SPSS 16.0 for Windows; SPSS Inc. Chicago, Illinois, USA).

Results

Clinicopathological profiles

The age of patients ranged from 22 to 35 years, and most of them were 30 years old. All patients manifested primary infertility. The size of the testis was within the normal range. The patients also presented a normal hormonal profile and azoospermia based on semen analysis (Table 1).

Histopathological evaluation

This study included 2 cases of normal spermatogenesis, 20 cases of moderate hypospermatogenesis, 11 cases of severe hypospermatogenesis, 25 cases of maturation arrest at the level of spermatids, 20 cases of maturation arrest at the level of secondary or primary spermatocytes, 5 cases of Sertoli cell-only syndrome or partial Sertoli cell-only syndrome, 2 cases of basement membrane hyalinization and peritubular fibrosis, and 1 case of tubular and peritubular sclerosis (Table 2).

Immunohistochemical measurement and expression

Mcl-1 expression was determined. In cases of normally appearing spermatogenesis (Fig. 1), strong positive reactions were observed in Leydig cells, moderate positive reactions in primary and secondary spermatocytes, and weak positive reactions in some spermatids and some spermatozoa. Cases of moderate hypospermatogenesis (No. 15) showed strong positive reactions in primary and secondary spermatocytes, negative reactions in the other stages of spermatogenesis, and positive reactions in Leydig cells (Figs. 2‒6). The other cases showed negative reaction in the tubules. In cases of severe hypospermatogenesis and maturation arrest, spermatocytes presented a negative reaction and Leydig cells were strongly positive (Figs. 7‒9). Cases with the Sertoli cell-only syndrome and peritubular fibrosis showed strong positive reactions in Leydig cells (Figs. 10 and 11, respectively). The two cases of Sertoli cell-only syndrome showed weak immunoreactivity in Sertoli cells but strong reactivity in Leydig cells (Figs. 12‒14, Table 3).

Discussion

Selection of subjects with azoospermia was based on the normal testis size and hormonal profiles and the absence of apparent genital tract obstruction. This selection aimed to minimize the need for testicular biopsy, which was limited for cases that may benefit from the procedures, particularly those who would undergo in vitro fertilization.

Apoptosis in testes is an important physiological mechanism that regulates the number of germ cells in the seminiferous epithelium. The Bcl-2 family of proteins mainly function in spontaneous apoptosis during normal spermatogenesis [ 12]. The growth, differentiation, death, and survival of spermatogonia are precisely regulated for proper production of spermatozoa [ 13]. Mice with a systemic deletion of the Mcl-1 gene died in the embryo stage because of implantation failure [ 14]. Previous studies indicated that Mcl-1 inhibited apoptosis, but the possibility that Mcl-1 can perform other non-apoptotic functions should be considered [ 15]. The current study investigated the immunohistochemical expression of Mcl-1 in testicular specimens. Strong immunoreactivity for Mcl-1 in Leydig cells was observed in all specimens under study. As Mcl-1 is an anti-apoptotic factor, almost no apoptosis occurred in these cells. Moderate positive reactions were also recorded in spermatocytes and spermatids, but these reactions were negative in few spermatozoa of subjects with apparently normal testes. Therefore, strong apoptotic activity occurred in these cells and may have caused the detected azoospermia despite the apparent normal spermatogenesis. Moderate reactivity was further detected in cases of hypospermatogenesis, which could be attributed to oligospermia. Negative reaction was commonly observed in cases of maturation arrest and germ cell aplasia (Sertoli cell-only syndrome, peritubular fibrosis, and sclerosis), which may explain the defective spermatogenesis in such cases. However, some cases of Sertoli cell-only syndrome showed moderate reactivity of Sertoli cells to Mcl-1, which may be attributed to the low activity of apoptosis in Sertoli cells and possibly caused by congenital or hereditary factors. Previous work on Mcl-1 expression in the testis reported weak reactions in all tubular cells [ 16]. In this study, Bcl-2 expression significantly differed and strong reactivity was noted in spermatocytes, particularly toward the lumen of the tubules [ 16]. Another study found negative reactions to Bcl-2 in all spermatocytes for cases of testicular varices [ 17]. Previous results further showed that spontaneous apoptosis occurred in all male germ-cell compartments [ 16]. The current results showed that apoptotic activity increased in these cells, which could be due to azoospermia.

In this study, two cases with normal spermatogenesis were detected among all cases of azoospermia. The results show that normal spermatogenesis does not exclusively suggest the obstruction of the duct system of the testis, but may also be attributed to the increased apoptosis inside the seminiferous tubules. In summary, cases of testicular biopsy should be stained with Mcl-1 to obtain apoptosis profiles for optimization of the diagnosis and effective management of male infertility.

Conclusions

The high occurrence of apoptosis (the rate of apoptosis increased more than that of proliferation) in seminiferous tubules may be due to azoospermia and male infertility. This very important aspect of spermatogenesis should be the focus of intensive research in the coming years. Further studies must be conducted particularly on knockout animal models with large sample sizes and with the use of other markers.

References

[1] Hetts SW. To die or not to die: an overview of apoptosis and its role in disease. JAMA1998; 279(4): 300–307

[2] Kerr JFR, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy. Cancer1994; 73(8): 2013–2026

[3] Brinkworth MH, Weinbauer GF, Bergmann M, Nieschlag E. Apoptosis as a mechanism of germ cell loss in elderly men. Int J Androl1997; 20(4): 222–228

[4] Dunkel L, Hirvonen V, Erkkilä K. Clinical aspects of male germ cell apoptosis during testis development and spermatogenesis. Cell Death Differ1997; 4(3): 171–179

[5] Hikim AP, Wang C, Lue Y, Johnson L, Wang XH, Swerdloff RS. Spontaneous germ cell apoptosis in humans: evidence for ethnic differences in the susceptibility of germ cells to programmed cell death. J Clin Endocrinol Metab1998; 83(1): 152–156

[6] Sinha Hikim AP, Swerdloff RS. Hormonal and genetic control of germ cell apoptosis in the testis. Rev Reprod1999; 4(1): 38–47

[7] Opferman JT, Iwasaki H, Ong CC, Suh H, Mizuno S, Akashi K, Korsmeyer SJ. Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science2005; 307(5712): 1101–1104

[8] Lipshultz LI, Howards SS. Evaluation of the subfertile male. In: Lipshultz LI, Howards SS. Infertility in the Male. 3rd ed. St. Louis: Mosby-Year Book, Inc., 1997: 173–193

[9] Coburn M, Kim ED, Wheeler TM. Testicular biopsy in male infertility evaluation. In: Lipshultz LI, Howards SS. Infertility in the Male. 3rd ed. St. Louis: Mosby-Year Book, Inc., 1997: 219–248

[10] Kovacs G. Assessment of the male partner. In: Kovacs G. The Subfertility Handbook: a Clinician’s Guide. Cambridge: Cambridge University Press, 2010: 54–59

[11] Boenisch T, Farmilo AJ, Stead RH, Key M, Welcher R, Harvey R, Atwood KN. Hand Book Immunochemical Staining Methods, 3rd ed. Carpinteria, California USA: DAKO Corporation, 2001: 1–62

[12] Kukner A, Oner J, Oner H, Ozan E. Effect of vitamin E on expression of Appoptosis Related Proteins in Immobilized Rat testes. J <?Pub Caret?>Anim Vet Adv2010; 9(1): 180–185

[13] Sugiyama N, Obinata M, Matsui Y. Bcl-2 inhibits apoptosis of spermatogonia and growth of spermatogonial stem cells in a cell-intrinsic manner. Mol Reprod Dev2001; 58(1): 30–38

[14] Rinkenberger JL, Horning S, Klocke B, Roth K, Korsmeyer SJ. Mcl-1 deficiency results in peri-implantation embryonic lethality. Genes Dev2000; 14(1): 23–27

[15] Yang T, Buchan HL, Townsend KJ, Craig RW. MCL-1, a member of the BLC-2 family, is induced rapidly in response to signals for cell differentiation or death, but not to signals for cell proliferation. J Cell Physiol1996; 166(3): 523–536

[16] Oldereid NB, Angelis PD, Wiger R, Clausen OP. Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis. Mol Hum Reprod2001; 7(5): 403–408

[17] El-Fackahany HM, Sakkas D. Abortive apoptosis and sperm chromatin damage. In: Zini A, Agarwell A. Sperm Chromatin: Biological and Clinical Applications in Male Infertility. New York: Springer-Verlag New York, 2011

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg