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The Use of Testicular FINE Needle Aspiration, Histology and Immunohistochemistry for Sertoli and GERM CELL Determination in Peri-Pubertal Bulls
Nicolas Negrin Pereira1, Pawel Borowicz1, Bryan Neville2, Jordan Flaten1 and Carl R. Dahlen.1
1Department of Animal Sciences, North Dakota State University, Fargo ND 58108.
2Central Grasslands Research Extension Center, Streeter ND 58483.
The relationship between the Sertoli cell population and number of germ cells within testicular tissue is a potential indicator of fertility in different mammalian species. Determining the population of Sertoli cells in peri-pubertal bulls may be a useful tool in identifying potential daily sperm production. Androgen receptors (AR) are expressed exclusively on Sertoli cells within the seminiferous tubule making them a potential target for Sertoli cell enumeration via immunohistochemistry. The objective of this study was to compare techniques of fine needle aspiration (FNA), histology, and immunohistochemistry to determine Sertoli and germ cell counts in the bull. Testicular parenchyma samples were obtained from 14 peri-pubertal Angus and Shorthorn bulls (287 ± 3.3 days of age, 310 ± 10 Kg). Smears for FNA were produced after collection with 22 gauge (FNA-F) and 16 gauge (FNA-G) needles, and tissues were cut for Hematoxylin/Eosin staining (HE) or incubated with mouse monoclonal antibody for androgen receptor immunofluorescence and counterstained with DAPI (ARIF). Pictures of HE stained specimens were assessed by manual count based on Sertoli cell morphology and ARIF were assessed by image analysis software ImagePro Premier based on AR positive vs counterstained cells. Germ to Sertoli cells ratio (GSR) was calculated dividing the total number of Germ cells by the total number of Sertoli cells within a seminiferous tubule section. Mean of all GSR were determined for each testicle and results of each technique were compared using the correlation procedure of SAS. Differences were considered significant when P<0.05. A positive correlation (r=0.58) was observed (P = 0.0015) between GSR obtained via HE and ARIF. In addition, a positive correlation (r=0.686) was observed between GSR obtained via FNA-F and FNA-G techniques (P = 0.001). No significant correlations were observed (P≥0.366), however, between GSR obtained via FNA (3.60 ± 0.47 and 3.59 ± 0.39 for FNA F and FNA G, respectively) and from tissue cuts (5.27 ± 0.41 and 4.44 ± 0.59 for HE and ARIF, respectively). The immunohistochemistry fluorescent method against AR we evaluated was a specific and novel tool for determining Sertoli cell populations in peri-pubertal bulls.
Key words: androgen receptors, Sertoli, techniques,