To investigate growth control mechanisms during spermatogenesis in vitro, [3H]thymidine incorporation into acid-insoluble macromolecules was used to quantify DNA synthesis in cultured spermatocysts (intact Sertoli cell/germ cell clones) derived from premeiotic (PrM), meiotic (M), and postmeiotic (PoM) regions of dogfish (Squalus acanthias) testis. Forty-eight hours after seeding in basal medium, DNA synthesis was > 7-fold higher in PrM cysts than in other stages, thus verifying the staging procedure. In autoradiograms, germ cells of PrM cysts (e.g., spermatogonial and preleptotene stages) were labeled all-or-none, but not all cysts were labeled, and later developmental stages (e.g., cysts with round or elongating spermatids) were never labeled. Fetal bovine serum (FBS, 10%) and insulin-transferrin-selenite (ITS, 10 micrograms-10 ng/ml) doubled DNA synthesis in PrM cyst cultures but had no effect at other stages. Bovine insulin (10 micrograms/ml) and human recombinant insulin-like growth factor-I (IGF-I, 15 ng/ml) also doubled [3H]thymidine uptake in PrM cultures, but lower doses were less effective and estradiol-17 beta, transferrin, adult shark serum, purified shark relaxin, and a variety of other known growth factors were neither stimulatory nor inhibitory at the doses and conditions tested. Sertoli cell monolayers derived from PrM- or M-stage spermatocysts displayed a dose-response increase in DNA synthesis after addition of IGF-I (15-75 ng/ml), with a maximal increment significantly greater than with 10% FBS. Using [3H]thymidine incorporation by PrM cysts as an end-point, stimulatory bioactivity was detected in the< 30,000 kDa fraction of spent media from PrM Sertoli cells, whereas the low molecular weight fraction of M-stage Sertoli cells was inhibitory. Gel electrophoretic analysis of the two fractions revealed qualitative and quantitative differences in protein banding patterns, reinforcing the view that secretory activity of Sertoli cells is stage related. Results of these studies implicate insulin/IGF-I in mechanisms governing proliferation of male germ cells and support the view that Sertoli cells have an autocrine or paracrine role as both targets and sources of growth regulatory factors.

In the dogfish sharkSqualus acanthias different germ cell stages are topographically segregated within the testis. Using this species we have developed methods for the isolation and culture of Sertoli cells from premeiotic, meiotic and post-meiotic stages of spermatogenesis and present preliminary evidence for stage-dependent variations in cell morphology and behavior, thymidine incorporation, protein synthesis and steroidogenesis. The goal of future studies is to determine how maturational changes are regulated in Sertoli cells and, in turn, to elucidate Sertoli cell-germ cell interactions.

The ultrastructure of large and small cells from corpora lutea of pregnant cows (days 45-280) were evaluated by electron microscopy. The distinguishing features of small cells (10-15 micron in diameter) included stacks of rough endoplasmic reticulum, whorls of smooth endoplasmic reticulum, elongated mitochondria containing crystalline-like inclusions, and cytoplasmic lipid droplets. The large cells (20-50 micron in diameter) contained numerous mitochondria packed tightly together (no elongated structures), an abundance of smooth endoplasmic reticulum (no whorls), and a large number of membrane-bound secretory granules (150-300 nm in diameter). These granules appeared to be packaged in the Golgi, accumulated at a paranuclear region, and migrated as a group to the cell membrane where they were exocytosed. These granules were first observed on day 45 and increased in number to reach a peak around day 200. Lipid droplets became a common cytoplasmic inclusion in the large cells during the third trimester of pregnancy. In addition, during this stage, an electron-dense substance began to accumulate in the mitochondria to such an extent as to occlude the cristae. These mitochondria looked like large (500-1800 nm) membrane-bound granules; however, they did not undergo exocytosis. Their appearance in large cells during the last 3 months of pregnancy may reflect a change in steroid metabolism. Thus, there are two morphologically distinct cell types throughout pregnancy in the cow. The large cell containing the secretory granules underwent what appeared to be a progressive state of apparent deterioration with advancing pregnancy. The morphology of the small cell did not undergo such a dynamic change. No morphological evidence was observed that would support a transition state between the two cell types.

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