Gene A. Hines

Sex steroid levels in the testes, ovaries, and pyloric caeca during gametogenesis in the sea star Asterias vulgaris

The concentrations of progesterone, testosterone, and estradiol were determined via radioimmunoassay in testes, ovaries, and pyloric caeca of the sea star Asterias vulgaris during one complete and two partial gametogenic cycles. These compounds were found in all tissues examined and were present in quantities similar to those reported previously in other echinoderms and in vertebrates. Testes and ovaries exhibited annual growth cycles during which testicular and ovarian mass increased up to 100-fold as gametes were produced and stored until spawning. Pyloric caecal mass varied during the annual reproductive season; however, no seasonal trends were apparent. In the testes, sex steroid levels were highest at the onset of spermatogenesis. Transient increases in the levels of estradiol coincided with spermatogonial mitotic proliferation. Transient increases in the levels of testosterone and progesterone in the testes coincided with spermatogenic column formation and with spermiogenesis, respectively. In the ovaries, estradiol and testosterone levels were highest at the onset of oogenesis while progesterone levels did not change significantly throughout the annual gametogenic cycle. Male and female pyloric caeca exhibited similar seasonal variations in levels of sex steroids as compared with the gonads. It is hypothesized that transient increases in the levels of sex steroids during gametogenesis may serve as endogenous modulators of reproduction.

Influence of insulin and testosterone on adrenocortical steroidogenesis in vitro: preliminary studies.

OBJECTIVE The mechanisms underlying the adrenal androgen (AA) excess of polycystic ovary syndrome (PCOS) remain unclear, although it is possible that the adrenocortical dysfunction may be a response to other, extraadrenal factors. Consistent with the pathophysiology of PCOS and with in vivo data in normal and PCOS women, we have hypothesized that insulin inhibits and that T stimulates AA secretion in vitro. DESIGN In vitro experimental study. SETTING University medical center. PATIENT(S) Normal human adrenals (n = 4 women, ages 25-57 years) were obtained with consent at the time of organ donation. INTERVENTION(S) Fresh adrenal tissue minces were incubated in serum-free medium with 10-microM pregnenolone substrate and 1-microM ACTH-(1-24). Challenge doses of 0.2, 1, 5, 20, and 100 nM of insulin and 1, 10, 100, 1,000, and 10,000 nM of T were added, and the media were sampled after 8 hours of incubation at 37 degrees C, 4% CO2. Dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEAS), and cortisol (F) were measured by radioimmunoassay (significant effects compared with the case of zero-dose control). MAIN OUTCOME MEASURE(S) The production of DHEA, DHEAS, and F in the media of the adrenal minces was compared between different subjects and at different concentrations of T and insulin. RESULT(S) Analysis of the combined data from all donors indicated that insulin stimulated DHEAS and suppressed DHEA production but had no consistent effect on F. Similar analyses of the combined data indicated that T had no significant predictable effect on the production of DHEAS, DHEA, or F. When examining donor data individually, insulin and T did elicit significant increases and/or decreases in steroid production within subjects, although no consistent trends were observed. CONCLUSION(S) On the basis of these data, it is clear that extra-adrenal factors such as insulin and T have some adrenal regulatory capacity. In general, insulin stimulated DHEAS and decreased DHEA production, suggesting that it increases adrenocortical sulfotransferase activity. However, although in the individual subjects studied, both insulin and T frequently altered the production of DHEAS, DHEA or F, these effects did not appear to be uniform or consistent from subject to subject. Expanded studies are required to confirm these results.

Altered autophosphorylation of the insulin receptor in the ovary of a woman with polycystic ovary syndrome

OBJECTIVE To determine whether the tyrosine autophosphorylation of the insulin receptor (IR) in the ovary of a woman with polycystic ovary syndrome (PCOS) was reduced compared to normal. DESIGN Experimental study. SETTING Tertiary care medical center. PATIENT(S) One woman with PCOS and one healthy control, both of whom underwent a hysterectomy and bilateral salpingo-oophorectomy. INTERVENTION(S) Plasma membrane fraction of ovarian tissue was isolated, and the IR was purified and concentrated. MAIN OUTCOME MEASURE(S) Western blots of the IR, which had been incubated with and without insulin, were prepared. Colorimetric and chemiluminescent methods were used to detect the presence of the IR beta-subunit and IR tyrosine autophosphorylation, respectively. RESULT(S) The presence of the beta-subunit of the IR was identified in both ovarian samples. The degree of insulin-stimulated IR tyrosine autophosphorylation, reflected by the mean (+/-SD) relative optical density of the 95 kd band, was 4.3-fold higher in the normal ovary compared to the PCOS ovary (0.56 +/- 0.18 optical density vs. 0.13 +/- 0.10 optical density, respectively). CONCLUSION(S) Tyrosine autophosphorylation of the IR may be decreased in the ovaries of women with PCOS, similar to the findings in other tissues. The mechanisms through which insulin acts to produce an excess in ovarian androgen production in the face of a decrease in ovarian IR autophosphorylation remain to be determined.

Examination of the chin or lower abdomen only for the prediction of hirsutism.

OBJECTIVE To test the hypothesis that scoring terminal hair growth on only the chin or abdomen can serve as a reliable predictor for hirsutism. DESIGN A prospective observational study. PATIENT(S) Six hundred and ninety-five consecutive hyperandrogenic women seen between June 1987 and December 1997. MAIN OUTCOME MEASURE(S) All hirsutism exams were performed by one examiner. Hirsutism was scored using a modification of the Ferriman-Gallwey (F-G) method. An F-G score of > or = 8 defined hirsutism. RESULT(S) Of the 695 women examined 352 (50.1%) had hirsutism scores of 8. Thirty percent (79 of 344) of women who had an F-G score of <8 had previously underwent electrology. If either the chin or lower abdomen hair growth score was > or = 2, the sensitivity was 100% for the prediction of hirsutism, although the specificity was 27%. The positive predictive value (PPV) for hirsutism using a hair score of > or = 2 at either of these sites was 58%. CONCLUSION(S) A hair growth score of > or = 2 on the chin or lower abdomen only was found to be a highly sensitive predictor for hirsutism. However, because of its very low PPV, this screening method is virtually useless in populations where the hirsutism frequency is expected to be low, about 5%. However, this screening method for the detection of hirsutism would be useful for the study of high-risk populations with an expected hirsutism prevalence of >20% (e.g., family studies).

Repeated freezing and thawing does not generally alter assay results for several commonly studied reproductive hormones

OBJECTIVE To determine the effect of repeated freezing and thawing, and storage temperature, on the assay results of commonly measured reproductive hormones, and sex hormone-binding globulin (SHBG) in human serum. DESIGN Prospective laboratory study. SETTING Academic medical center. PATIENT(S) Four men and three pregnant women in the third trimester. INTERVENTION(S) Pooled serum from men and pregnant females were frozen at either -20 degrees C or -70 degrees C. Aliquots were then subjected to repeat freeze/thaw cycles, from 1 to 10 times, and assays were performed after the final freeze/thaw cycle. MAIN OUTCOME MEASURE(S) Assay results for eight hormones (FSH, LH, PRL, androstenedione (A), 17alpha-hydroxyprogesterone, P, insulin, and SHBG, as a function of the number of freeze/thaw cycles and storage temperature. RESULT(S) Only SHBG in male serum at -20 degrees C and P in pregnant serum at -70 degrees C showed statistically significant decreases in assay results with repeated freeze/thaw cycles (3.3% and 1.1% per cycle, respectively). All other analytes did not show significant changes as a function of freeze/thaw cycles or storage temperature. CONCLUSION(S) There is no consistent or predictable alteration in the results of SHBG, or the glycoprotein and steroid hormones evaluated, as a function of repeated freeze/thaw or the storage temperature of human serum.

Androgen metabolism in somatic and germinal tissues of the sea star Asterias vulgaris

1. Cell-free homogenates of male and female pyloric caeca, body wall, testis and ovary were incubated with radiolabeled 3H-androstenedione. 2. Pyloric caeca had highest rates of androstenedione conversion. The predominant metabolites in the pyloric caeca were testosterone, 5 alpha-androstane-3 beta, 17 beta-diol and 5 beta-androstane-3 beta, 17 beta-diol. 3. In body wall, testicular and ovarian homogenates, androstenedione was converted primarily to testosterone and also to 5 alpha-androstanedione and epiandrosterone. 4. Qualitative and quantitative differences in androgen metabolism in somatic and germinal tissues may be related to tissue-specific regulation of cellular metabolism.

Sex Steroid Extraction from Echinoderm Tissues

Abstract A method for sex steroid extraction from selected echinoderm tissues is presented. This method utilizes solvent and solid based extraction techniques and is reliable for small tissue samples. Tissue homogenates were incubated in warm acidic conditions prior to organic extraction of steroids. Further isolation of steroids using Sep-Pak cartridges and a simple elution scheme resulted in percent recoveries ranging from 56%-90% depending on steroid and tissue type.

Levels of progesterone, testosterone, and estradiol, and androstenedione metabolism in the gonads of Lytechinus variegatus (Echinodermata:Echinoidea)

Levels of progesterone (P4), testosterone (T) and estradiol (E2) indicated significant variation among individual echinoids during the annual cycle, reflecting generally the variation in gamete development that can be observed among individuals. Testosterone and E2 levels in both the ovaries and testes were higher during the period of gonadal growth. Levels of all steroids were greatly reduced compared to those levels reported for asteroids. Differences in the levels of P4, T, and estrogens between asteroids and Lytechinus variegatus may be related to differences in gonad morphology and nutrient storage capacity between asteroids and echinoids. It was hypothesized that the low levels of steroids detected in L. variegatus reflect paracrine-like mechanisms in cell signaling as compared to endocrine-like mechanisms proposed to be involved in regulating gonad function in asteroids. Both the ovaries and testes of L. variegatus had the capacity to synthesize T and a variety of 5alpha-reduced androgens including 5alpha-androstane-3beta,17beta-diol and 5alpha-androstane-3alpha,17beta-diol (5alpha-adiols) from androstenedione (AD) in 8 h. Estrogen synthesis was not detected. The sex-specific pattern of accumulation of 5alpha-adiols in the ovaries and testes suggests that the 5alpha-adiols may affect processes related to reproduction in L. variegatus.