Frank Eertmans

Development and validation of a high-performance liquid chromatography (HPLC) method for the determination of human serum albumin (HSA) in medical devices

Because of its nutritional, anti-oxidative, and cryoprotective properties, human serum albumin (HSA) is an important ingredient of the culture and cryopreservation media for assisted reproductive techniques (ART) such as in vitrofertilization (IVF) and intracytoplasmic sperm injection (ICSI) procedures. Several tools are available for the determination of this serum protein in biological samples and pharmaceutical preparations, including colorimetric, electrophoretic, and immunological assays. However, because of inter-assay variability and accuracy problems of the above-mentioned assays, we have chosen to develop and validate a reverse phase (RP) high-performance liquid chromatography (HPLC) method to assess HSA content in ART-related media. Briefly, a gradient elution (a combination of acetonitrile/water, supplemented with 0.1% (v/v) trifluoroacetic acid) was used to separate samples on a C4 (n-butyl-coated silica) column. Two main peaks were observed at 4.970 and 8.715 min, representing the stabilizer N-acetyl-tryptophan (N-Ac-Trp) and HSA respectively. Validation of the method demonstrated that HSA can be determined in an accurate and precise manner, in a range between 0.4 and 25 mg ml−1, without interference of matrix ingredients. The limit of detection (LOD) and lower limit of quantification (LLOQ) values were 0.128 and 0.386 mg ml−1, respectively. In summary, this RP-HPLC method serves as a quality control for ART product release and stability studies. If required, the method can be easily adapted for assessment of HSA in biological samples and pharmaceutical preparations.

Single Cell and Spheroid Collagen Type I Invasion Assay

Tumor invasion is the outcome of a complex interplay between cancer cells and the stromal environment and requires the infiltration of a dense, cross-linked meshwork of collagen type I extracellular matrix. We use a membrane-free single-cell and spheroid-based complementary model to study cancer invasion through native collagen type I matrices. Cell morphology is preserved during the assays allowing real-time monitoring of invasion-induced changes in cell structure and F-actin organization. Combination of these models with computerized quantification permits the calculation of highly reproducible and operator-independent data. These assays are versatile in the use of fluorescent probes and have a flexible kinetic endpoint. Once the optimal experimental conditions are empirically determined, the collagen type I invasion assays can be used for preclinical validation of small-molecule inhibitors targeting invasion. Initiation and monitoring of the single-cell and spheroid invasion model can be achieved in 8 h (over 3 days) and in 14 h (over 5 days), respectively.

Estrogen receptor alpha (ERα) and insulin-like growth factor I receptor (IGF-IR) cross-talk in the gonadotropic αT3-1 cell line

In reproductive tissues such as the breast and the uterus, cell proliferation and differentiation is strongly regulated by complex interactions between estrogen receptor α (ERα) and growth factor receptors. In the present study, we investigated the potential occurrence of such cross‐talk in the murine, gonadotropic αT3‐1 cell line, which expresses ERα and the IGF‐I receptor (IGF‐IR). Under estrogen‐free conditions, basal cell proliferation and ER‐mediated gene transcription was strongly inhibited by the selective estrogen receptor modulator (SERM) 4‐hydroxy‐tamoxifen (4‐OH‐Tam) and by the pure anti‐estrogen ICI 182,780 (ICI). These effects can be reversed by either 17‐β‐estradiol (E2) or insulin‐like growth factor I (IGF‐I), both exerting modest mitogenic effects in the αT3‐1 cell line. Furthermore, IGF‐I enhanced both basal and E2‐induced ER‐driven gene transcription. This may be explained, at least in part, by enhanced phosphorylation of ERα at serine 118, a prerequisite for the transactivation capacity of the receptor. Finally, the IGF‐I‐induced response on cell growth and ER‐mediated transactivation can be inhibited with either ICI or 4‐OH‐Tam. In conclusion, our data indicate IGF‐IR and ER interactions in the αT3‐1 cell line, an in vitro model for the pituitary gonadotrophs, hereby suggesting a role of IGF‐I in the regulation of gonadotropin synthesis and secretion. J. Cell. Physiol. 212:583–590, 2007.

Estrogen receptor signaling is an unstable feature of the gonadotropic LβT2 cell line

The murine, gonadotropic LbetaT2 cell line was assessed as a potential in vitro model to analyze estrogen receptor (ER)-mediated regulation of luteinizing hormone (LH) synthesis and secretion. In agreement with limited literature data, repeated exposure to (sub) physiological concentrations of gonadotropin-releasing hormone enhanced LHbeta-subunit gene expression, being the rate-limiting step of LH synthesis, and the corresponding LH secretory response. However, in the same subclone of the LbetaT2 cell line, we observed that LH production was not affected following exposure to E(2), which is in contrast to previously reported weak or modest effects. One explanation may be the absence of measurable ERalpha protein expression on the one hand and impaired ER signal transduction on the other. Furthermore, an alternative ERalpha mRNA splicing variant was detected in the LbetaT2 cell line, which (theoretically) encodes for a protein that may alter ERalpha transcriptional activity, depending on the cellular context. The studied LbetaT2 subclone did not show a generalized impairment of nuclear receptor function, as we observed androgen- and glucocorticoid-induced gene transcription, together with enhanced LH secretory response following dexamethasone treatment. Since its development, the gonadotropic LbetaT2 cell line served as a reference model to study gonadotroph-specific effects because of its mature properties. Nevertheless, this cell line does not seem to be a suitable in vitro model for the study of estrogenic regulatory effects at the level of the pituitary gonadotrophs in view of the unstable nature of ER signaling in LbetaT2 cells.

Male Reproductive Status and Its Relationship With Man-Made, Hormone-Disrupting Substances: Studies in Flanders, Belgium

Sperm quality seems to have declined over time in certain regions in the world. This regional trend coincides with the progressive increase in the incidence of male genital tract anomalies including testicular maldescent, hypospadias and testicular cancer. The association of these pathologies has been called the ‘testicular dysgenesis syndrome’ (TDS). The possible implications of decreased male fertility cannot be underestimated. Couples attending fertility clinics have a higher chance of multiple pregnancies, preterm delivery and consequently also of low birth weight, which is a known risk factor for a number of pathological conditions at adulthood. Many studies suggest TDS to be caused by man-made endocrine disrupters, mostly chemicals initiating estrogen actions (xenoestrogens) or inhibiting the effects of endogenous androgens (anti-androgens). Thousands of man-made chemicals have been released into the environment in vast quantities since the chemical industry began to boom in the 1950’s. Humans and wildlife are exposed to these chemicals through their nutrition, the air, the water and, more importantly, through the placenta during foetal development. In order to reliably estimate the reproductive health risks posed by these chemicals, scientific programmes should pursue the identification of the routes via which humans and wildlife are exposed to these xenobiotics using biomarkers of internal and external exposure. These are new and important tools, complementary to the traditional chemical analytical techniques as they reflect the integrated response of an individual or test organism to multiple compounds, accounting for possible additive, antagonistic or synergistic effects.

Further evaluation of the biological activity of the unique gonadotropin-releasing hormone peptide in the guinea pig brain.

In this study we compared the biological activity of a unique form of gonadotropin-releasing hormone (GnRH) in the brain of the guinea pig (gpGnRH) with mammalian GnRH (mGnRH). In gpGnRH, the highly conserved histidine in position 2 (His(2)) and leucine in position 7 (Leu(7)) are substituted by tyrosine and valine, respectively. The gpGnRH was less potent than mGnRH in stimulating the release of luteinizing hormone (LH) in vivo in the guinea pig and displayed only low activity in the rat. The gpGnRH was more rapidly degraded by serum proteolytic enzymes than mGnRH. It is concluded that gpGnRH displays lower biological activity than mGnRH in both rat and guinea pig, which may be due in part to its greater susceptibility to proteolytic degradation besides differences in receptor affinity and/or activation.

Difference in receptor-binding contributes to difference in biological activity between the unique guinea pig GnRH and mammalian GnRH

In the current study, we compared the in vitro potency of a unique form of gonadotropin-releasing hormone (GnRH) present in the brain of the guinea pig (gpGnRH) with that of mammalian GnRH (mGnRH) as well as their binding affinities to the GnRH receptor. In gpGnRH, the highly conserved histidine in position 2 (His(2)) and leucine in position 7 (Leu(7)) are substituted by tyrosine and valine, respectively. In vivo, gpGnRH was shown to be less potent than mGnRH, possibly in part because of higher susceptibility to enzymatic degradation. In the present in vitro experiments, we observed that gpGnRH was less potent than mGnRH in stimulating the release of luteinizing hormone (LH) from primary pituitary cell cultures of the rat, and at lower concentrations from primary pituitary cell culture of the guinea pig, too. These results were confirmed by radioligand-binding studies. It is concluded that the lower biological activity of gpGnRH in both rat and guinea pig may be explained by the difference in binding to the target cells, although additional factors such as proteolytic degradation may also contribute to the observed phenomenon.

An Improved Neutral a-Glucosidase Assay for Assessment of Epididymal Function—Validation and Comparison to the WHO Method

Neutral α-glucosidase (NAG) activity in human seminal plasma is an important indicator for epididymis functionality. In the present study, the classic World Health Organization (WHO) method has been adapted to enhance assay robustness. Changes include modified enzyme reaction buffer composition and usage of an alternative enzyme inhibitor for background correction (glucose instead of castanospermine). Both methods have been tested in parallel on 144 semen samples, obtained from 94 patients/donors and 50 vasectomized men (negative control), respectively. Passing-Bablok regression analysis demonstrated equal assay performance. In terms of assay validation, analytical specificity, detection limit, measuring range, precision, and cut-off values have been calculated. These data confirm that the adapted method is a reliable, improved tool for NAG analysis in human semen.