The granulosa cell produces a protein inhibitor of aromatase activity (follicle-regulatory protein: FRP), which recently was purified to homogeneity. To determine the possible involvement of FRP in follicular maturation, we examined the size distribution of follicles and their morphological patterns as well as serum steroid levels after the systemic administration of FRP and/or gonadotropin to guinea pigs, which have 5-6 days between luteolysis and ovulation in a 16-day cycle. FRP was partially purified from porcine follicular fluid by ammonium sulfate precipitation (0-35%), Dye Matrex Orange A Chromatography, dialysis, and lyophylization. To investigate the effect of pregnant mare's serum (PMS) during the periovulatory period in follicular development, adult guinea pigs underwent unilateral ovariectomy on Days 10, 12, and 14 of the estrous cycle (N = 6 each). Guinea pigs were injected twice daily with vehicle or PMS (5 IU) and 2 days thereafter the remaining ovaries were removed. Another group of guinea pigs received, in addition, intraperitoneal injections of FRP (1 mg) each morning from Day 8 of estrus until they were killed. The resected ovaries were fixed, embedded in paraffin, serially sectioned (7 micron), and stained with Azan for comparative study via light microscopy. All follicles greater than 400 micron were classified by size, and the atretic pattern was determined by mural granulosa cell pyknosis and antral sloughing. The distribution of follicular size was not affected by hemicastration at Day 10, although the percentage of total atretic follicles decreased. In the PMS-treated group, there was a significant decrease in the number of viable follicles (700-899 micron) after hemicastration. Also pronounced in follicles of this size was the lack of mid-atretic follicles. After injections of FRP for 3 or 5 days, the overall number of follicles was almost doubled as compared to the number found in the normal ovary. Additionally, there was a significant increase in the percentage of follicles that were recently atretic, although the total percentage of atretic follicles was unchanged. After hemicastration at Day 10 followed by FRP treatment for 2 days, the total percentage of atretic follicles in the remaining ovary decreased to 18% compared with 35% in the normal ovary, 46% in the hemicastrated plus PMS-treated group, and 38% in the hemicastrated and PMS- and FRP-treated group (all p less than 0.01). Treating the hemicastrated animal with PMS increased the percentage of atretic follicles in all groups.(ABSTRACT TRUNCATED AT 400 WORDS)
We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome. The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2 n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2 n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2 n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.
The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monodonal antibody or polydonal antisera and then incubated with either biotinylated mouse 1gM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small-or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamma; the antral granulosa cells ofviable follides did not stain. Neither primordial follides nor pre-antral foffides (<300 im in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas
A protein from follicular fluid [referred to as follicle regulatory protein (FRP)] which inhibits aromatase activity in granulosa cells was recently isolated and partially characterized. The purified FRP was used to produce a monoclonal antibody which was used to develop an enzyme-linked immunosorbant assay suitable for quantitation of FRP in urine. Twelve normal premenopausal women underwent daily collection of blood and first morning urine samples, beginning on the 1st day of menses, as well as daily ultrasonographic evaluation of follicular diameter, beginning on the 10th day of the menstrual cycle, until the onset of the next menses. Serum estradiol, progesterone, LH, and FSH levels were determined by RIA. Urinary FRP levels increased in the midfollicular phase, reached their zenith in the midluteal phase [mean, 0.38 +/- 0.03 (+/- SE) immunoreactive units; 1 immunoreactive unit = approximately 1 ng FRP/mL.mg creatinine], and then declined to reach their nadir (not detectable) during the early follicular phase. Immunohistochemical evaluation of ovarian tissue demonstrated that anti-FRP localized to mural granulosa cells in viable follicles, to all follicular epithelial cells in atretic follicles, and to the large cells of the corpus luteum. These findings indicate that immunoreactive FRP levels in urine change during the menstrual cycle and suggest a relationship among FRP, follicular maturation, and corpus luteum formation.
Evaluation of Recon-mended Methods for Radioisotope Red Cell Survival Studies. Tohoku J. exp. Med., 1976, 120 (3), 219-223•\Mean red cell lifespan in normal subjects and in patients with various hematological disorders was examined with 51Cr and DF32P. The results with 51Cr were corrected for 51Cr elution using
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