In the ovary, follicle-stimulating hormone (FSH) binds selectively to its own receptor (FSHR) exclusively localized on granulosa cells, resulting in activation of adenosine 3?,5?-cyclic monophosphate (cAMP)-dependent and other cAMP-independent signalling pathways. As a result of these signal transductional pathways, FSH induces changes in the intracellular Ca²⁺ concentration of granulosa cells which represent a major ovarian cell type that utilizes Ca²⁺ release to stimulate steroidogenesis. However, the exact mechanisms responsible for these changes are not fully elucidated at present. Several studies suggest that the changes in intracellular Ca²⁺ levels after FSH binding to the cell may be due to an influx of calcium through FSHR-regulated Ca²⁺ channels. In spite of the consistent ability of FSH to mediate Ca²⁺ influx in both testicular Sertoli cells and ovarian granulosa cells, the intracellular targets of the varying second messenger signals are not fully understood. Nevertheless, there is now a great deal of experimental evidence that in many cell types (inclusive granulosa cells) inositol 1,4,5-trisphosphate receptors (IP₃Rs), which have Ca²⁺ channel activity, are responsible for the inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release from intracellular stores. Three distinct types of IP₃Rs have been identified, termed IP₃R-1, IP₃R-2, and IP₃R-3, and the expression of different IP₃Rs has been confirmed in the mammalian reproductive system (ovaries, oocytes, ova) of many species, including mouse, hamster, cow, and human. Most of the studies deal with the presence and different distribution patterns of IP₃R-1 during maturation and fertilization of oocytes. IP₃R-1 was found to be highly expressed in oocytes of Graafian and mature follicles, mediating most of the Ca²⁺ release during fertilization. Other studies provided evidence that IP₃R-2 is present in follicular granulosa cells, whereas IP₃R-3 is less abundant than IP₃R-2. Due to the high-affinity binding of IP₃ to IP₃R-2 and the reported abundance in granulosa cells of other species, it is thus of special interest to determine the expression of IP₃R-2 in porcine ovaries. To detect cell-type-specific IP₃R-2 expression, we performed immunohistochemistry on paraffin sections of methanol–glacial acid-fixed ovaries at different stages of the oestrous cycle and reverse transcription-polymerase chain reaction (RT-PCR).

The gilts of the German Landrace were obtained from the experimental station of animal husbandry, animal breeding, and small animal breeding of the University of Hohenheim. Oestrous detection was performed twice daily in the presence of a boar. The animals ranging in age from 7 to 11 months were slaughtered on days 3–6 (early luteal phase, n=5), 11–13 (mid-luteal phase, n=4) and 18–20 (follicular phase, n=3) after the onset of oestrous (first day of standing OESTROUS=day 0). Immediately after slaughter, the genital tracts were removed and explored for confirmation of ovarian status and for signs of pathological alterations. The ovaries were subsequently cut into halves, fixed in methanol/glacial acid (2:1, 24 h), and paraffin embedded according to standard procedures. For immunohistochemical staining, serial sections were cut at a thickness of 5 ?m and mounted on superfrost^® glass slides.

Total RNA was isolated from follicles of different cycle stages and from luteal tissue of the early and mid-luteal phase using a RNA isolation kit (Invitek, Berlin, Germany, Cat.-No. 10602003). To rule out contamination by genomic DNA, isolated RNA was treated with RNAse-free DNAse according to the manufacturer’s protocol (Promega, Mannheim, Germany). Thereafter, RNA was quantified by measuring the absorbance at 260 and 280 nm and further, its quality was checked by electrophoresis using a 20% polyacrylamide gel and stained with a silver-staining kit (Amersham, Freiburg, Germany). For performing the reverse transcription, 1–2 ?g total RNA and a mixture according to the manufacturer’s protocol (Stratagene, Amsterdam, The Netherlands) were used in a reaction volume of 20 ?l. The tubes were incubated at 37°C for 60 min followed by additional 5 min at 95°C and chilled on ice. The primers used in this study for the PCR were derived from the known genomic DNA sequence of the human inositol 1,4,5-trisphosphate receptor type 2 (IP₃R-2) forward (nucleotide 857–876), 5?-ATGCGTGTGTCCTTGGATGC-3?, reverse (nucleotide 1226–1245), 5?-GTAGCAGAAGTAG-CTGATTG-3?. For the PCR, a 50 ?l volume was used containing 2 ?l of cDNA, 1 ?l of each primer (50 pmol forward+50 pmol reverse), 5 ?l 10× buffer (100 mM Tris-HCl pH 8.8, 500 mM KCl, 15 mM MgCl₂, 1% Triton X-100), 1 ?l dNTP mixture (10 mM each), 2.5 ?l 100% DMSO and 1 unit DyNAzyme DNA polymerase (Biometra, Göttingen, Germany). In a Techne Genius Thermocycler (Thermo-Dux, Wertheim, Germany), we used a denaturation step at 94°C for 2.5 min, followed by 36 cycles at 94°C for 1 min, specific annealing temperature at 65°C for 1 min and 72°C for 2 min and for 10 min in the last cycle. Finally, the PCR amplification products were visualized after electrophoresis on a 20% polyacrylamide gel using a DNA silver-staining kit (Amersham). Measurement of the existing PCR amplification product delivered the value of the expected size (388 bp).

Deparaffinized sections were heated in a microwave in 10 mM sodium citrate buffer at pH 6.0 (4×5 min at 600 W) to expose antigenic sites. Sections were then rinsed 3 times in phosphate-buffered saline (PBS, pH 7.4) and incubated with 1% hydrogen peroxide in double-distilled water for 10 min at room temperture (RT) to inhibit endogenous peroxidase activity. After rinsing in PBS, specimens were treated with 10% normal rabbit serum blocking solution (Dako, Hamburg, Germany) for 30 min at RT. Sections were incubated overnight in a humid chamber at 4°C with anti-IP₃R-2 diluted 1:200 in PBS (Santa Cruz Biotechnology, Heidelberg, Germany). The next day, sections were rinsed and treated with the biotinylated rabbit-anti-goat immunoglobulin (diluted 1:400 in PBS) for 30 min at RT (Dako). After a final PBS wash and incubation with streptavidin–biotin–horseradish-peroxidase complex for 30 min at RT (Strept-ABC kit; Dako), the reaction product was visualized with 0.05% 3,3? diaminobenzidine hydrochloride (DAB) and hydrogen peroxide (0.6×10^?3%) in 0.1 M PBS (Biotrend Chemicals, Köln, Germany). Finally, sections were counterstained with Mayer’s haematoxylin, dehydrated, cleared with xylene, and mounted in DePeX (Boehringer, Ingelheim, Germany). Using a bright-field light microscope (DMRBE; Leica, Bensheim, Germany), and a video camera (ProgRes; Kontron Instruments, Watford, UK) coupled to a Pentium PC (Fujitsu Siemens; Munich, Germany), the intensity of immunohistochemical staining of IP₃R-2 was graded on a scale from unstained (?) to strong staining (+++). Immunohistochemical controls were performed by (1) replacing the primary antibody with nonimmune serum, (2) omitting the secondary antibody and (3) incubation with DAB solution alone to ensure specificity of staining. Specimens from bovine oviduct were used as positive controls for IP₃R-2 immunostaining.

In order to test whether expression of the IP₃R-2 protein was paralleled by corresponding expression of the mRNA, RT-PCR was performed on RNA extracted from the same samples taken at different stages of the oestrous cycle. When amplified with the primers common to IP₃R-2, a RT-PCR product of the expected size (approximately 388 bp) was clearly detected in the early luteal and follicular phase of the oestrous cycle. However, no detectable PCR product was found in the corpus luteum of the mid-luteal phase of the oestrous cycle.

Figure 1. Expression of IP₃R-2 in porcine follicles and corpora lutea at different stages of the oestrous cycle. Representative RT-PCR amplification products of the expected size of 388 bp are shown in ovarian samples collected in the early luteal (n=10) and follicular phase (n=6) of the oestrous cycle, respectively. Ovarian luteal tissue RNA in the mid-luteal phase (n=8) is devoid of any corresponding band. Lane 1, molecular size marker (1 kb); lane 2, early luteal phase (day 3); lane 3, mid-luteal phase (day 13); lane 4, follicular phase (day 20); lane 5, no-template control with IP₃R-2 primers.