Chicken embryo extract mitigates growth and morphological changes in a spontaneously immortalized chicken embryo fibroblast cell line.

The SC-1 spontaneously immortalized chicken embryo fibroblast (CEF) cell line has been established recently. Although this cell line had been in culture for over 3 yr, its growth rate has remained lower than that of primary CEF cells, and the morphology has not been as uniform as observed in primary cells. In the present study, the SC-1 cell line was treated with chicken embryo extract (CEE) to determine whether growth rates could be increased and cell morphology enhanced. The CEE also was tested on primary CEF cells, another spontaneously immortalized CEF cell line (DF-1), and on 2 other nonvirally and nonchemically immortalized CEF cell lines (BCEFi and HCEFi). Results indicated that concentrations of CEE > or = 100 microg/mL inhibited growth of all cells tested. However, addition of 50 microg of CEE/mL enhanced the growth rate and improved the morphology of the SC-1 cells. Addition of CEE to the other immortal or primary CEF cells did not increase the growth rate or change their morphology. Analysis of mRNA expression revealed that SC-1 cells treated with 50 microg of CEE/mL had lower levels of the p16(INK4a) alternate reading frame sequence (ARF) and E2F-1 than untreated SC-1 cells. The increased growth rate and improved morphology of the SC-1 cells achieved with CEE treatment were retained following removal of CEE, and these improvements should aid in increasing the utility of the SC-1 cell line as a cellular/molecular reagent.

Induction of ovarian cystic follicles in sheep.

Cystic follicles are a significant cause of infertility in women, dairy cattle and sheep. Sheep were used as a model to identify factors that may elicit formation of cystic follicles. Insulin resistance and elevated LH activity were tested in overweight ewes because of associations among these factors and the formation of cystic follicles. Sheep were synchronized using a progesterone-releasing pessary and insulin resistance was induced during the synchronization period through administration of bovine somatotropin. Following removal of pessaries follicular growth was stimulated by treatment with eCG or eCG and hCG (PG-600). Follicular growth was monitored via daily transrectal ultrasonography and blood samples were collected for hormonal analyses. Six of 18 ewes had a subnormal or absent preovulatory gonadotropin surge and developed cystic follicles. Neither insulin resistance nor elevated LH activity were associated with formation of cystic follicles. Ewes that developed cystic follicles were heavier (93 +/- 4 kg) than ewes that ovulated (81 +/- 3 kg; P = 0.02). Furthermore, following pessary removal and initiation of daily ultrasonography, ewes that developed cystic follicles lost body weight (-3 +/- 1%), while ovulatory ewes continued to gain body weight (1 +/- 1%; P = 0.005). It is speculated that in heavy ewes metabolic factors associated with acute body weight loss inhibit the positive feedback of estradiol and thereby suppress the preovulatory gonadotropin surge leading to formation of cystic follicles.

Inhibin localization in equine granulosa-theca cell tumours and inhibin forms in tumour fluid.

The aim of this study was to examine inhibin production in granulosa-theca cell tumours (GTCT). The experimental aims were: (i) to determine GTCT cell types that produce inhibin alpha- and betaA-subunits; (ii) to determine whether alpha- and betaA-subunit forms differ in GTCT fluid and normal equine follicular fluid (eFF); and (iii) to determine whether dimeric inhibin (alpha betaA) is present in GTCT plasma and tumour fluid. Plasma, tumour fluid and tumour tissue were collected from mares (n=6) with GTCT. Plasma and eFF were collected during the follicular phase from mares (n=4) undergoing normal cycles. Immunohistochemical examination of GTCT tumour sections showed strong inhibin alpha- and betaA-Subunit immunostaining in granulosa cells and polyhedral-shaped cells in the thecal-stromal layer. The presence of polyhedral-shaped cells was related to testosterone concentration in tumour fluid. Low molecular weight alpha-subunit forms were less abundant in tumour fluid than in eFF, whereas the amounts of betaA-subunit forms were similar in tumour fluid and eFF. Concentrations of betaA were increased in plasma from mares with GTCT and similar in tumour fluid and eFF. In summary, lower molecular weight alpha-subunit forms were less prominent in GTCT fluid than in eFF and concentrations of betaA were higher in GTCT plasma than in control plasma.

Dimeric inhibin concentrations in mares with granulosa-theca cell tumors.

OBJECTIVE: To determine whether concentrations of dimeric inhibin (CaCA) are greater in plasma and tumor fluid from mares with granulosa-theca cell tumors (GTCT), compared with concentrations in plasma and equine follicular fluid (eFF) from control mares. ANIMALS: 6 mares with GTCT and 12 clinically normal mares. PROCEDURE: The alphabetaA immunoradiometric assay used 2 antibodies, one against each subunit of inhibin (alpha and betaA subunits). Tumor tissue, tumor fluid, and a single blood sample were collected at the time of surgical removal of the GTCT. A single blood sample was collected from 7 control mares during various stages of the estrous cycle. Five other control mares were ovariectomized when their ovaries contained growing follicles of 25 to 35 mm in diameter. A blood sample and eFF from the largest follicle were collected at the time of ovariectomy. RESULTS: Mares with GTCT had significantly greater plasma concentrations of betabetaA (mean +/- SEM, 0.86 +/- 0.53 ng of recombinant human-alphabetaA/ml), compared with control mares (0.14+/-0.02 ng/ml). Concentrations of alphabetaA in tumor fluid and eFF were similar. Concentrations of alphabetaA were significantly lower after ovariectomy. CONCLUSIONS AND CLINICAL RELEVANCE: Dimeric inhibin concentration was higher in plasma from mares with GTCT than in plasma from control mares. Increased granulosa cell mass and loss of mechanisms regulating alphabetaA release in mares with GTCT likely accounted for the increase in plasma concentrations. Measurement of alphabetaA concentrations may be useful for identifying mares with GTCT.