Date of Award

1-1-1978

Embargo Period

1-1-1978

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Anatomy

College

College of Graduate Studies

First Advisor

R. R. Markwald

Second Advisor

Richard Dom

Third Advisor

William J. Dougherty

Fourth Advisor

S. S. Spicer

Fifth Advisor

Elsie Taber

Sixth Advisor

Jerome G. Ondo

Abstract

Sertoli cells were collected from adult (200-300 gms.) and young (21 days old) Wistar rats and cultured under various conditions. “Young” Sertoli cells (cells obtained from 21-day-old rats) were maintained in Medium 199, with and without 10% fetal calf serum (10% FCS), and collected every 24 hours following plating for up to two weeks. Ultrastructurally, freshly isolated cells were similar to in situ cells of the same age. They retained this phenotype over the incubation period, indicating failure to differentiate structurally commensurate with comparable aged in vivo cells. Positive staining for succinic dehydrogenase (SDH), lactic dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) indicated that in vitro cells remained viable in terms of cytosolic and mitochondrial metabolism. However, relative enzyme staining patterns of cells cultured for 10 days (chronologically 31 days old) did not conform to those of 31-day-old in vivo cells but remained identical to freshly collected cells. Although follicle stimulating hormone (FSH-NIAMD-B1; 50μg or 150μg/2ml medium) affected cell shape and stimulated cellular metabolism, indicated by elevated LDH staining, it did not induce ultrastructural maturation. Persistent high ratio of LDH/SDH further indicated lack of in vitro cellular differentiation in the presence of FSH. Presence or absence of 10% FCS mimicked presence or absence of FSH. In summary, young Sertoli cells in vitro failed to keep pace morphologically or cytochemically with in vivo Sertoli cells of the same chronological age. FSH and/or 10% FCS did not override “arrested” differentiation. “Adult” Sertoli cells (cells obtained from adult rats) were maintained in Medium 199, with or without 10% FCS, and collected every 24 hours following plating. Ultrastructurally, freshly isolated cells were similar to in situ cells of the same age. They degenerated structurally, however, and by 10 days looked similar to cells in advanced stages of hydropic degeneration. Marked decrease in metabolic enzyme staining further indicated cellular degeneration, whereas loss of staining for 3β-hydroxysteroid dehydrogenase (3βOLDH) indicated cellular dedifferentiation. Neither the presence of FSH, 10% FCS nor a combination of FSH and 10% FCS prevented this pattern of progressive decrease in viability and death of adult Sertoli cells in culture. When co-cultured with peritubular cells, adult Sertoli cells remained metabolically viable judged by enzyme histochemistry. Ultrastructurally, cells incubated for two weeks looked similar to in vivo cells of the same age, except for a decrease in the amount of smooth endoplasmic reticulum (SER). This decrease correlated with lack of staining for SER-bound 3βOLDH. At the ultrastructural level there was an extracellular material similar in size and structure to collagen which bridged Sertoli cells and peritubular cells. It appeared to be secreted by peritubular cells which developed the typical ultrastructural profile of protein-secreting cells. Isolated adult Sertoli cells did not survive in long term culture, whereas those co-cultured with peritubular cells did. Presumably peritubular cells (and/or their collagen-like extracellular product) provided a microenvironmental factor conducive to maintenance of adult Sertoli cells in vitro in a state similar to in situ Sertoli cells of the same age. FSH potentiated Sertoli cell spreading in co-culture but did not restore 3BOLDH staining nor SER in amounts typical of adult cells.

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