In a recent report we demonstrated that a high (micromolar) concentration of progesterone (P) specifically down-regulates LH receptor (R) expression and function in murine Leydig tumor cells. The aim of the present study was to characterize further the putative novel R, mediating these P effects in the murine Leydig tumor cell line, mLTC-1. The binding of [3H]P to these cells revealed a high (Kd, approximately 9.3 nmol/liter) and a low affinity (Kd, approximately 284 nmol/liter) component, and the binding displayed with specificity (P > dehydroepiandrosterone > 17-OHP). The binding was apparently different from that of the classical nuclear PR in the following ways. 1) The P/glucocorticoid antagonist RU 486 did not compete with [3H]P binding to the mLTC-1 cells. 2) No expression of the classical PR messenger RNA was detected, despite clear P binding to these cells, by Northern hybridization or RT-PCR. 3) An antibody against the C-terminal end of the classical PR (alpha c-262) revealed in mLTC-1 cells several molecular size protein bands between 45-57 kDa on Western hybridization, whereas these immunoreactive proteins were faintly recognized by another antibody (alpha-PR) directed toward the NH2-terminal region of the classical PR. The sizes of the immunoreactive molecules were relatively similar to those detected using the same antibodies in human sperm lysates, but were at variance with the classical PR (120, 94, and 60 kDa), detected with these antibodies in human uterus. The immunoreactive proteins bound peroxidase-labeled-P, which could be displaced in the presence of a 10-fold excess of free P. 4) An immediate increase in the intracellular free calcium level was observed after P treatment in cultured mLTC-1 cells, whereas it also increased the 45Ca2+ entry within 15 min in these cells. 5) Increasing doses of P (0.1-10 micromol/liter) demonstrated significant inhibition of LH receptor messenger RNA levels in a dose-dependent manner in mLTC-1 cells. In conclusion, a nonclassical PR is expressed and functional in these cells, and it is clearly distinct from the classical nuclear PR. It is apparent that recently reported inhibitory effects of P on LH receptor gene expression and function are mediated through this novel type PR in mouse Leydig cells.
We describe a luminescence immunoassay for measuring transferrin in human seminal plasma. Human transferrin conjugated with 7-[(N-4-aminobutyl)-N-ethylamino]naphthalene-1,2-dicarboxylic acid hydrazide was used to monitor the immunological reaction. The conjugate was stable for at least one year. The sensitivity (2 ng per tube) of the assay allows measurement of this protein in diluted seminal plasma. Results by this method (y) correlated well (r = .9681) with those by a conventional RIA method (x): y = 1.000x + 1.646 micrograms per ejaculate. Seminal transferrin concentrations are reported for normal control subjects, vasectomized subjects, and infertile patients. The method described appears suitable for measurement of seminal transferrin as an index of Sertoli cell function in male infertility.
Study question Could Reactive Oxygen Species (ROS) detected in human spermatozoa represent a predictive marker of fertilization ability? Summary answer ROS detected by CellROX® Orange probe is related to a better sperm quality and function, indicating the sensitivity of the probe to identify physiological ROS. What is known already Oxidative stress (OS), defined as an unbalance between ROS production and antioxidant defenses, is one of the causes of male infertility. A small amount of ROS is necessary for the physiological sperm function, however high ROS levels could impair fertility potential inducing damages at membrane, protein and DNA levels. Previous studies performed by using different methods and probes for OS evaluation in semen or in spermatozoa highlighted the negative role of ROS on sperm functions. However, such studies were not conclusive because of the small number of included subjects and of high variability in the cohorts. Study design, size, duration Observational study conducted on 73 male partners of infertile couples attended consecutively to the Andrology Laboratory of Careggi University Hospital of Florence from September to December 2021. Analyses were performed on both washed and Swim-up selected spermatozoa. Participants/materials, setting, methods After routine semen analysis, washed and Swim-up selected spermatozoa were incubated with CellRox®Orange (a fluorescent probe able to reveal OS in viable cells), at the concentration of 1 µM for 30 minutes at 37°C, 5%CO2, and revealed by flow cytometry. Sperm DNA fragmentation (by TUNEL/PI method), sperm kinematic parameters and hyperactivated motility (by C.A.S.A. system) were also assessed. In some samples a double staining with CellRox®Orange and Annexin V (which stains phosphatidylserine externalization) was performed. Main results and the role of chance We found that the percentage of spermatozoa positive to CellRox® Orange is positively correlated with routine seminal parameters. Although this result appears in contrast with most studies in literature reporting negative correlations between OS and semen parameters, it can be explained by the fact that the probe is specific for viable spermatozoa characterized by a better motility and morphology. Considering the importance of distinguishing between positive and negative ROS in spermatozoa, we further investigated the significance of ROS detected by this probe. To understand whether the probe marks spermatozoa with a better quality, CellRox® Orange positivity was evaluated in Swim-up selected spermatozoa finding significantly higher levels of CellRox® Orange positivity respect to unselected samples. Furthermore, the fact that most of the CellRox® Orange positive spermatozoa were negative for Annexin V (which reveals cells with early signs of apoptosis) is a further confirmation of the good quality of CellRox® Orange positive spermatozoa. Another evidence is represented by the finding that we observed a negative correlation between OS detected by CellRox® Orange and sperm DNA fragmentation, although several studies have shown a positive relationship between these two parameters (when OS is measured by different probes). Limitations, reasons for caution Up to now, the study involved a limited number of subjects. Further experiments should be performed to increase the number of subjects in order to confirm the results. Wider implications of the findings Results of this study together with those previously published evidence that different OS is detected depending on which probe is used. CellRox® Orange seems to be sensible for physiological ROS detection, therefore, it could be employed in future studies aimed to evaluate the association between OS and assisted reproduction outcomes. Trial registration number not applicable
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