Androgens are essential in males for the development and maintenance of specific reproductive organs such as testes, prostate, and epididymis. The biochemical aspects of androgen metabolism are now of special interest since recent data imply that estrogens play an important role in modulating the function of the male gonad, especially in germ cell development. The formation of estrogens from androgens depends on functionally active aromatase, a microsomal enzyme (also known as estrogen synthase, P450arom, EC 1.14.14.1), that has been demonstrated in both Leydig and Sertoli cells, and recently in germ cells of various animal species.

The mammalian Y chromosome plays a crucial role in sex determination and fertility of males. Over the last decade, various Y-chromosome microdeletions have been identified in human infertile patients with azoospermia or oligozoospermia. In mice, the Y-del mutation is caused by partial deletion in the long arm of the Y chromosome. This mutation was found during routine chromosomal analysis of B10.BR males in The National Institute of Genetics (Mashima, Japan). Since 1987, these mutants have been bred at the Department of Genetics and Evolution of Jagiellonian University (Kraków, Poland). They have been extensively studied with respect to sperm quality and their fertilization capacity. In comparison with the B10.BR strain, its congenic mutant strain is characterized by increased numbers of abnormal spermatozoa and low fertilization efficiency as demonstrated in vitro. In mutant males with the deletion in the long arm of the Y chromosome, a high percentage of sperms with abnormal heads has been observed, and among them class 1b, which is characterized by flattening of the acrosomal cap, is predominant. Moreover, formation of the acrosome during spermiogenesis is altered in mutant mice. Other important testicular parameters have also been analyzed such as testis weight, proportion of abnormal tubules, number of Sertoli cells, number of pachytene spermatocytes, and number of step-9 spermatids. Because spermatozoa of control and mutant mice are qualitatively and quantitatively different, we decided to compare expression of aromatase as well as steroidogenic activity in testes of both strains, especially because aromatase is a key enzyme in the modulation of balance between sex-steroid hormones.

Bishop et al (1985) identified a mouse Y-derived sequence, Y353/B, which detects multiple copies of related sequences on the mouse Y long arm. The exact function of Y353/B-related sequences is yet unknown, but it has been demonstrated that it is part of an expressed region, and a strong candidate to contain the Smy gene. Later, tested the presence of Y353/B-related sequences in mutant males with Y-chromosome deletions.

Southern-blot analysis was performed on DNA from B10.BR-Y^del mice and the congenic B10.BR strain to test copy numbers of the Y353/B-related sequence. Furthermore, testes of these two strains of mice, B10.BR and B10.BR-Y^del, respectively, were examined using immunohistochemistry, Western-blot analysis, and radioimmunological assays, to localize aromatase, and to measure steroid hormone levels, respectively.

Animals were bred at the Department of Genetics and Evolution of Jagiellonian University. All mice were given commercial pelleted diet, water ad libitum, and maintained under a 12 h light–dark cycle. Thirty-five mature males (2–3 months old) were used for the experiments. They were killed by cervical dislocation. Testes were used for either isolation of Leydig cells and in vitro cultures, or routine histology. Furthermore, immunocytochemistry on cultured cells or immunohistochemistry on testicular sections was applied.

Experiments were performed in accordance with Polish legal requirements, under the licence provided by the Commission of Bioethics at Jagiellonian University.

Genomic DNA was isolated from the tail. DNA (20 ?g of each sample) was restricted with EcoRI, electrophoresed on a 1% agarose gel, and transferred onto nitrocellulose membranes (Hybond C; Amersham, Buckinghamshire, UK). Membranes were hybridized with ³²P-labeled Y353/B probe (Rediprime II Kit, [-³²P] dCTP, Amersham;) at 65 °C, overnight. After hybridization, membranes were washed at high stringency (0.2×SSC at room temperature for 30 min, 0.1×SSC, 0.1% SDS at 65 °C for 2×1 h) and exposed overnight at ?70 °C. To check the integrity of DNA and its quantity, the membranes were stripped and re-hybridized with Zfy-derived probe. These experiments were repeated 4 times.

Eight decapsulated testes from each mouse strain were used for the preparation of Leydig cell suspensions according to the procedure previously described in detail. Briefly, crude suspensions of Leydig cells were obtained by short digestion (10 min, at 37°C) using trypsin in PBS (0.25%), and then they were serially sieved through steel meshes with pore sizes of 156 and 74 ?m, respectively (US Standard Sieve series with ASTME 11 Specifications; Dual MFG, Chicago, IL, USA). Supernatants were collected and centrifuged at 180g for 5 min. The resulting cell pellets were washed twice and subjected to purification by centrifugation on a continuous 10–90% (v/v, 50 ml) Percoll gradient (Pharmacia, Uppsala, Sweden) using a method described by with some modifications. After centrifugation at 800g for 25 min at 4°C, because the low temperature significantly prevented cell aggregation, the cell bands containing Leydig cells were collected, washed, and centrifuged once or twice at room temperature using low-speed centrifugation (90g for 10 min). Leydig cells were cultured for 48 h in 24-well culture dishes (Nunc, Kalmstrup, Denmark) in Eagle’s medium supplemented with 2% calf serum and -glutamine (Laboratory of Sera and Vaccines, Lublin, Poland). Penicillin (120 iu/ml) was also added. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ in air. The purity of the cells was approx. 87% and was checked by a histochemical assay of ?⁵, ?-hydroxysteroid dehydrogenase (?⁵, ?-HSD) activity. Viability of the cells as assessed by the trypan blue exclusion test was over 95%. Purity and viability of the cells were determined in triplicate. For immuncytochemical purposes, each well was closed with a round coverslip with an appropriate diameter. Culture media were collected and stored at ?20° for hormone measurements.

It has been previously reported that spermatozoa of B10.BR-Y^del mice possess a cytoplasmic droplet attached to their flagella. Therefore, only spermatozoa of this strain were isolated to demonstrate the presence of aromatase in cytoplasmic droplets. Sperm cell isolation has been described in detail. Small drops of suspensions of sperm cells were transferred to slides precoated with poly-lysine and smears were carefully made. Immunocytochemistry was applied to detect aromatase in cytoplasmic droplets (see below).

Testes of five animals of both strains were fixed in 4% paraformaldehyde and embedded in paraplast (Monoject Scientific Division of Scherwood Medical, St. Louis, MO, USA). Sections (6 ?m thick) were mounted on slides coated with 3-aminopropyl-triethoxysilane (APES; Sigma, St. Louis MO, USA), deparaffinized, and rehydrated. To optimize immunohistochemical staining, sections were immersed in 10 mM citrate buffer (pH 6.0) and treated in a microwave oven (2×5 min, 600 W) for high-temperature antigen unmasking. Testicular sections were stained 4 times and staining was scored for the presence and intensity of immunostaining. Aromatase staining was designated as absent, weak, moderate, or strong on the basis of visual examination of cytoplasmic localization of the antigen.