Steve D. Holmes

Transferrin and gonadal dysfunction in man

Transferrin concentrations were quantitated in the seminal fluid of normal, oligozoospermic, and azoospermic patients and related to other known parameters of testicular function. Transferrin concentration in the semen of patients 2 months after vasectomy (13.2 +/- 1.8 micrograms/ml) was significantly less than that obtained from pregnancy-proven donors (65.6 +/- 10.1 micrograms/ml). This indicates that approximately 80% of the seminal fluid transferrin is derived from the testes. The concentration of transferrin in semen from patients with azoospermia (14.4 +/- 1.8 micrograms/ml), severe oligozoospermia (17.5 +/- 1.7 micrograms/ml), and moderate oligozoospermia (21.8 +/- 4.3 micrograms/ml) was significantly lower than normospermic groups. Serum follicle-stimulating hormone (FSH) was measured in a group of infertile patients; those having an elevated FSH had a significantly lower concentration of semen transferrin, 14.1 +/- 1.6 micrograms/ml, compared with patients who had FSH levels within the normal range (33.7 +/- 5.3 micrograms/ml). It is possible that the underlying cause in decreased spermatogenesis associated with both an elevated FSH and a decreased transferrin concentration is impaired Sertoli cell function.

Peritoneal fluid plasminogen activator activity in endometriosis and pelvic adhesive disease

Plasminogen activator activity in PF was assayed by the fibrinolysis method described by Strickland and Beers. In 45 patients studied, there were no discernible differences according to whether patients had endometriosis and/or pelvic adhesive disease. No differences were detected according to when in the menstrual cycle the sample of PF was obtained. These data are in concordance with a previous report and taken together suggest that there is no difference in fibrinolytic mechanisms in PF in patients with or without endometriosis and/or pelvic adhesive disease, when compared with control subjects. If such differences exist, they may be present in the tissues, per se, but are not discernible in PF.

Purification of a progesterone receptor from rabbit uterus

Abstract A progesterone receptor has been purified to homogeneity from rabbit uterus by steroid affinity chromatography. The receptor was obtained in 5% yield, with a specific activity for [3H]progesterone binding of 14,580 pmol/mg protein. The pure receptor migrated as a single band on SDS-polyacrylamide electrophoresis, with a MW of 70,000. Progesterone binding to the receptor was heat labile and was displaced by an excess of R5020. Photoaffinity labeling of the pure receptor with [3H]R5020 corresponded to the major photoaffinity labeled species in crude cytosol.

Rapid method for quantitation of androgen binding protein in Sertoli cell cultures and its use for measurement of binding kinetics.

The accurate measurement of the kinetics of binding of 5 alpha-dihydrotestosterone to the Sertoli cell specific protein, androgen binding protein (ABP), has been frustrated by the extremely rapid rate of dissociation of the ABP-dihydrotestosterone complex. We describe a rapid and highly sensitive assay suitable for ABP quantitation which utilizes DEAE Bio-Gel and [3H]dihydrotestosterone. The assay has been used to accurately measure the rate of dissociation (8.25 X 10(-4) s-1, t1/2 14 min) and the rate of association (2.04 X 10(5) M s-1) of the binding of [3H]dihydrotestosterone to rat ABP. The ratio of these rate constants is in perfect agreement with the equilibrium dissociation constant determined by Scatchard analysis (4.0 nM). This multipoint assay is extremely rapid such that binding can be measured at equilibrium, it has high precision (coefficient of variation 3%), and is particularly useful at low protein concentrations (50 ng/ml); furthermore, the assay background of nonspecific 3H-binding is extremely low (0.2%). Since at such low protein concentrations a 10 point Scatchard analysis can be performed on 1 ml culture medium containing as little as 3 fmol ABP, the assay is suitable for monitoring changes in ABP secretion resulting from manipulations of cells in culture. The assay which utilizes DEAE Bio-Gel A is compared to five alternative methods: the standard method of steady state gel electrophoresis, Dextran-coated charcoal assay, hydroxylapatite assay, DEAE filter assay, and radioimmunoassay. The DEAE Bio-Gel assay has advantages over all of these alternative methods. In summary, this new assay is particularly useful for monitoring temporal changes in the secretion of ABP, and the method is equally effective in quantitating ABP in rat, rabbit and hamster Sertoli cell cultures.

Effect of Cannabinoids on Human Sertoli Cell Function In Vitro

Exposure to marijuana adversely affects spermatogenesis in humans and rodents. Since the Sertoli cell interfaces between changes in the serum hormonal milieu and the adluminal compartment and acts in various ways to support the germinal epithelium, this adverse marijuana mediated effect might be transmitted locally in the testes of humans by interference with Sertoli cell function. This report describes the effect of three active marijuana components: delta-9-tetrahydrocannabinol (THC), cannabidiol (CD) and cannabinol (CN) on various human Sertoli cell markers: transferrin, protein secretion and gamma glutamyl transpeptidase activity utilizing cultured human Sertoli cells. THC, CD, and CN tested in concentrations up to 200 ng/ml did not change transferrin secretion by these human Sertoli cells. Transferrin represents 1-4% of the total newly synthesized proteins of human Sertoli cells in culture. Even when the Sertoli cells were incubated for 30 days with THC there was no effect on transferrin secretion. The presence or absence of follicle-stimulating hormone, testosterone (T) or Fe3+ in the culture media did not modify the effect of THC. THC, CD, and CN also did not alter total protein secretion by Sertoli cells nor affect the level of gamma glutamyl transpeptidase activity.

Unique Characteristics of Rat Sertoli Cell Secreted Growth Factor

The Sertoli cell is believed to play an important role in the regulation of spermatogenesis. Therefore, there has been considerable interest in Sertoli cell secreted proteins. Recently, our laboratory has shown that rat Sertoli cell conditioned medium (SCCM) contained a potent mitogenic activity, Sertoli cell secreted growth factor (SCSGF).’ We have further characterized this activity. Sertoli cells were isolated and cultured from 19-21-, 35-, and 45-day-old rat^.^.^ The SCCM was used in a cell growth assay with A431 cells (a human epidermoid carcinoma cell line). Subconfluent A431 cells were plated in Dulbecco’s modified Eagle’s medium (DME) with 5% fetal bovine serum (FBS) on 30 x I5 mm petri dishes. After 24 h, the medium was removed by aspiration and 1 ml of DME and 0.5-1.0 ml of SCCM added; control dishes received DME only. After 3 d incubation, the cells were removed by trypsin/EDTA and counted in a Coulter counter. All samples were assayed in triplicate. The ability of 50% SCCM to stimulate the growth of A431 cells was tested in the presence of no serum, 0.5% FBS, 2% FBS, and 5% FBS with three applications over a 6 d period (TABLE 1). In the absence of serum there was a 3.6-fold stimulation of A431 cell growth by day 6. In 0.5% FBS and 50% SCCM, A431 cells were maximally stimulated to grow with a 7.2-fold stimulation. Even in the presence of 2% or 5% FBS, there was a 2.3-fold stimulation of A431 cell growth with 50% SCCM. The secretion of the mitogen was age-dependent. The SCCM prepared from 35or 45-day-old rats had nearly twice the proliferative effect of SCCM from 1921-day-old rats. Furthermore, SCCM was stimulatory not only to A431 cells, but also for DDT’MF-2 cells, Swiss and BALB/c 3T3 cells, and R3327H-G8-AI cells.’ In an attempt to classify the SCSGF, we asked what other classes of growth factors stimulate the growth of A431 cells. Although EGF (2.5 ng/ml) inhibited the growth of A431 cells by about 50%, TGF, (2 ng/ml), TGF, (2 ng/ml), TGF, (2 ng/ ml), bombesin (10 ng/ml), FGF (10 ng/ml), somatomedin C (33.3 ng/ml), and PDGF (12.5 ng/ml) were ineffective alone and did not potentiate the stimulation seen with SCCM. Transferrin ( 5 pg/ml) and lactate (0.65 mM), both secreted by Sertoli cells, did not stimulate A431 cell growth. The mitogenic activity is acid-stable (pH 3.0, 1 h, room temperature), does not bind to heparin-agarose, is stable to freeze-thaw, is resistant to treatment with RNase A ( I 0 0 pg/ml, 2 h, room temperature) (TABLE 2), and has a molecular weight of 6-8000. These results, however, do not preclude the possibility that a

Delta-9-tetrahydrocannabinol stimulates ABP secretion from rat sertoli cells in vitro

The effect of delta-9-tetrahydrocannabinol (THC) on rat Sertoli cell function was investigated. THC significantly increased ABP secretion by 1.5- to 2.1-fold but did not consistently enhance the stimulation of ABP induced by FSH, testosterone or dibutyryl cyclic AMP. ABP was measured by steady-state polyacrylamide gel electrophoresis, DEAE Bio-Gel and immunoassay; all three methods gave similar results. The minimal concentration of THC that stimulated ABP was 10 ng/ml; maximal stimulation was observed with 100-200 ng/ml. This effect was specific since THC did not affect gamma glutamyl transpeptidase activity or the secretion of plasminogen activator, lactate and transferrin. This observation that THC affects ABP secretion specifically is the first report of any differential effect of a drug on Sertoli cell secretion.

Ion exchange, chromatofocusing and size exclusion high-performance liquid chromatography of the human uterine progesterone receptor

The human uterine progesterone receptor was subjected to high-performance liquid chromatography on size exclusion, anion exchange and chromatofocusing columns. For the rapid isolation of the receptor, recovery of [3H]progesterone as well as protein from the columns was essential. The size exclusion columns (G2000 SW and G3000 SW) as well as Mono P HR 5/20 chromatofocusing column adsorbed [3H]progesterone and thus were not useful for separation purposes. The anion exchange (polyanion SI-17) and chromatofocusing columns, AX500, and IEX 540 DEAE gave very good recoveries of protein (greater than 90%) and [3H]progesterone; 80, 66 and 88% respectively. These columns gave rapid and reproducible separation of the progesterone receptor from other cytosol proteins.