Canadian HealthCare News

Characterization of Rabbit Kitl cDNA The nucleotide sequence for the coding region of rabbit Kitl was 91%, 90%, 89%, and 89% identical at the nucleotide level to human, mouse, rat, and bovine Kitl, respectively. The deduced amino acid sequence of the cDNA was 86%, 86%, 84%, and 84% identical to human, mouse, rat, and bovine KITL, respectively (see Supplemental Fig. 1). These data indicate substantial interspecies conservation at gene and protein levels. (more…)

A considerably higher proportion of follicles in rabbit ovarian cortical explants degenerated in culture compared with follicles in mouse ovaries, possibly due to the extensive dissection undertaken to generate rabbit ovarian cortical explants which was not required for the preparation of mouse ovaries for culture. It is also possible that the culture system employed did not adequately support the development of later-stage rabbit follicles. However, given that at the initiation of culture the majority of follicles were in the primordial and early primary stage, whereas at the end of the culture period healthy multilayered growing follicles were often observed, we suggest that the growth and development of early rabbit follicles in vitro was supported by this culture system. A definite role for KITL in later follicle development would need to be addressed by the use of an isolated follicle rather than the explant culture system described above. (more…)

Primary follicles were those surrounded by a complete layer of cuboidal granulosa cells. Growing primary follicles had multiple layers of granulosa cells along one side and a single layer on another. Preantral follicles had two or more complete layers of granulosa cells in the absence of an antrum. Small antral follicles had patchy fluid-filled zones between the granulosa layers, whereas in large antral follicles the fluid patches had merged into a single large cavity. Antral follicles were not observed in cultured ovaries and explants. (more…)

In every third section follicles were counted and scored for stage of development, and the diameter of each oocyte was measured across the largest cross-section. Of follicles deemed healthy, only those with a visible germinal vesicle were counted or measured. The mean diameter of germinal vesicles for the mouse ranged from 9.6 6 0.01 im to 19.8 6 2.6 im (primordial to preantral follicles), and for the rabbit from 19.2 6 0.01 im to 22.2 6 0.03 im (primordial to primary follicles). (more…)

Both the recombinant mouse KITL protein and neutralizing antibody were obtained from Sigma Chemical Co. Doses of recombinant mouse KITL were selected on the basis of concentrations previously shown to promote oocyte growth and follicle activation in the mouse and rat, respectively. The dose of neutralizing antibody was determined using the ED50 provided by the manufacturer. In similar experiments rabbit ovarian explants were incubated with 150 ng/ml of recombinant rabbit KITL (see Supplemental Table 2). (more…)

Ovaries and explants were cultured on collagen I- and fibronectin-coated nitrocellulose membranes (0.45-im pore size; Millipore Corp., Bedford, MA) and mounted on triangular metal grid supports in Falcon Organ Culture Dishes (Becton Dickinson Co., Franklin Lakes, NJ). Coating was performed by incubating the membranes in 0.75 mg/ml collagen and 0.1 mg/ml fibronectin for 1 h at 37°C. Ovaries and ovarian explants were cultured in groups of three to five for 8 days in Dulbecco Modified Eagle medium (DMEM)/Ham F12 1:1 (v/v) (GibcoBRL, MD) supplemented with 10% fetal bovine serum, 0.03 IU recombinant FSH (Gonal-F 75; Serono, Italy), 5 ig/ml insulin, 5 ig/ml transferrin, 5 ng/ml selenium, and 0.1 iM retinoic acid. (more…)

The tissue sections (four from each ovary) were deparaffinized in xylene and then rehydrated in alcohol. Pretreatment, hybridization, and posthybridization washes were performed according to protocols described in the Non-radioactive In Situ Hybridization Application Manual (Roche Diagnostics). Kitl sense and antisense probes were hybridized overnight at 50°C. Kit sense and antisense probes were hybridized overnight at 68°C. Probes were detected immunohistochemically using the DIG Nucleic Acid Detection Kit (Roche Diagnostics) according to protocols described by the manufacturer. (more…)

The tissues were then incubated with biotin-conjugated goat anti-rat immunoglobulin G (IgG; Sigma Chemical Co.) diluted 1:1000 in PBS for 1 h at 37°C. Antibody binding was visualized using the ImmunoPure ABC Peroxidase Staining Kit (Pierce, Rockford, IL) and the Liquid DAB-Plus Substrate Kit (Zymed, San Francisco, CA). Sections were counterstained with hematoxylin and visualized by light microscopy using an Olympus BH-2 microscope. Negative controls, in which the primary antibody was replaced with preimmune serum, were routinely performed on adjacent serial sections, and no specific staining was observed. (more…)

Two booster injections of protein (50 ig), dissolved in PBS (50 il) and emulsified in an equal volume of Freunds Incomplete Adjuvant, were given at 2-wk intervals, commencing 4 wk after priming injections. Fourteen days following the final boost, rats were anesthetized with carbon dioxide and then exsanguinated by cardiac puncture. Blood sera were collected and tested for specificity against the respective immunogens and commercially obtained protein (recombinant mouse SCF; Sigma Chemical Co.) by Western blot (not shown). (more…)

PCR primers, plasmids, and bacteria used for cloning and recombinant protein expression are described (see Supplemental Table 2, available online at: http:// www.biolreprod.org/). Recombinant proteins were affinity purified with Ni-NTA resin (Qiagen, Australia) and renatured by dialysis using protocols described in The QIAexpressionist (Fifth edition, Qiagen; http://www1.qiagen. com/literature/handbooks/PDF/Protein/Expression/QXP_QIAexpressionist/ 1024473_QXPHB_0603.pdf). (more…)