A remaining problem in male reproduction is the exact relationship between steroidogenesis and spermatogenesis. In mammals it is difficult to obtain successive spermatogenic stage for biochemical analysis, thus we have chosen for study species in which (1) germ cells in different stages of maturation are segregated topographically within the testis (2) Leydig cells, Sertoli cells or Sertoli/germ cell units (follicles) are readily separated from other testis components; (3) morphological and physiological changes in the testis are a normal part of seasonal reproductive cycles (dogfish, salamander, squirrel). To date, we have documented stage-dependent variations in steroidogenic enzymes; steroid receptor number, occupancy and acceptors; and a novel sex steroid binding protein. We have also described the morphological correlates of these functional changes. Preliminary data show a pathway of communication from more mature to less mature stages and a role for steroids as parahormones. Techniques developed for culturing staged Sertoli/germ cell units (follicles) and staged Sertoli cells open new possibilities for studying the spermatogenic progression, Sertoli- germ cell interactions, and direct effects of steroids. Using light and electron microscopy; autoradiography; immunocytochemistry; enzyme tracer analysis; radioimmunoassay; receptor analysis; and cell culture techniques, we will: (1) Determine whether Sertoli cells alone account for stage-dependent steroidogenesis; (2) Determine whether stage-dependent changes in microsomal enzymes accurately reflect endogenous steroid production; (3) Describe the basic morphological and functional characteristics of cultured Sertoli cells from early, mid- and late stages; observe effects of time, germ cells and a normal 3-D configuration; (4) Measure steroidogenesis in Sertoli cells of a seasonal rodent during the annual cycle and localize P-450 enzymes by immunocytochemistry; (5) Pinpoint cellular targets of steroid action in the zoned testis using autoradiography; (6) Characterize steroid receptors in a seasonal rodent; measure changes during spermatogenesis; (7) Quantify germ cell/Sertoli cell proliferation, survival and differentiation in response to steroids in vitro; (8) Evaluate spermatogenesis in vivo after discrete intratesticular steroid placement; (9) Advance collaborative efforts to isolate testicular aromatase and use antibodies to study intratesticular distribution and molecular relatedness of this enzyme through evolution. Studies in this laboratory show the feasibility of using animal models outside the range of common laboratory species to advance materially our understanding of testicular endocrinology.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD016715-11
Application #
3313871
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1981-12-01
Project End
1993-11-30
Budget Start
1991-12-01
Budget End
1993-11-30
Support Year
11
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Boston University
Department
Type
Schools of Arts and Sciences
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Callard, G V; Jorgensen, J C; Redding, J M (1995) Biochemical analysis of programmed cell death during premeiotic stages of spermatogenesis in vivo and in vitro. Dev Genet 16:140-7
Piferrer, F C; Callard, G V (1995) Inhibition of deoxyribonucleic acid synthesis during premeiotic stages of spermatogenesis by a factor from testis-associated lymphomyeloid tissue in the dogfish shark (Squalus acanthias). Biol Reprod 53:390-8
Barry, T P; Thomas, P; Callard, G V (1993) Stage-related production of 21-hydroxylated progestins by the dogfish (Squalus acanthias) testis. J Exp Zool 265:522-32
Cuevas, M E; Collins, K; Callard, G V (1993) Stage-related changes in steroid-converting enzyme activities in Squalus testis: synthesis of biologically active metabolites via 3 beta-hydroxysteroid dehydrogenase/isomerase and 5 alpha-reductase. Steroids 58:87-94
Dubois, W; Callard, G V (1993) Culture of intact Sertoli/germ cell units and isolated Sertoli cells from Squalus testis. II. Stimulatory effects of insulin and IGF-I on DNA synthesis in premeiotic stages. J Exp Zool 267:233-44
Callard, G V (1992) Autocrine and paracrine role of steroids during spermatogenesis: studies in Squalus acanthias and Necturus maculosus. J Exp Zool 261:132-42
Cuevas, M E; Callard, G (1992) In vitro steroid secretion by staged spermatocysts (Sertoli/germ cell units) of dogfish (Squalus acanthias) testis. Gen Comp Endocrinol 88:151-65
Singh, S; Callard, G (1992) Identification of an androgen receptor in the zonal testis of the salamander (Necturus maculosus). Gen Comp Endocrinol 86:220-30
Cuevas, M E; Callard, G (1992) Androgen and progesterone receptors in shark (Squalus) testis: characteristics and stage-related distribution. Endocrinology 130:2173-82
Cuevas, M E; Miller, W; Callard, G (1992) Sulfoconjugation of steroids and the vascular pathway of communication in dogfish testis. J Exp Zool 264:119-29

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