Carlos Zumoffen

Oviductal secretion and gamete interaction

Experimental evidence from the last 30 years supports the fact that the oviduct is involved in the modulation of the reproductive process in eutherian mammals. Oviductal secretion contains molecules that contribute to regulation of gamete function, gamete interaction, and the early stages of embryo development. The oviductal environment would act as a sperm reservoir, maintaining sperm viability, and modulating the subpopulation of spermatozoa that initiates the capacitation process. It could also contribute to prevent the premature acrosome reaction and to reduce polyspermy. Many studies have reported the beneficial effects of the oviductal environment on fertilization and on the first stages of embryo development. Some oviductal factors have been identified in different mammalian species. The effects of oviductal secretion on the reproductive process could be thought to result from the dynamic combined action (inhibitory or stimulatory) of multiple factors present in the oviductal lumen at different stages of the ovulatory cycle and in the presence of gametes or embryos. It could be hypothesized that the absence of a given molecule would not affect fertility as its action could be compensated by another factor with similar functions. However, any alteration in this balance could affect certain events of the reproductive process and could perhaps impair fertility. Thus, the complexity of the reproductive process warrants a continuous research effort to unveil the mechanisms and factors behind its regulation in the oviductal microenvironment.

Proteins from human oviductal tissue-conditioned medium modulate sperm capacitation

BACKGROUND Spermatozoa acquire the ability to fertilize an oocyte when they become capacitated. Capacitation takes place when sperm pass through the female reproductive tract, interacting with female fluids. Both tyrosine phosphorylation of sperm proteins and the ability to respond to acrosome reaction (AR) inducers have been associated with sperm capacitation. Recent data indicate that conditioned media (CM) from human oviductal tissue culture decrease sperm affinity for the zona pellucida in vitro. Since capacitation enables the sperm-oocyte interaction, the aim of the present study was to investigate the effect of CM on events related to sperm capacitation and to assess whether these effects were permanent. METHODS Oviductal tissue was obtained from premenopausal patients (scheduled for hysterectomies because of uterine fibromyoma). The tissues were cultured as explants and CM were collected. Explant viability was assessed as tissue DNA integrity. Normozoospermic semen samples were obtained from healthy donors. Motile spermatozoa were incubated under capacitating conditions with or without increasing protein concentrations of CM for 6 or 22 h. Human follicular fluid-induced AR was detected by the Pisum sativum technique. Tyrosine phosphorylated proteins were detected with a monoclonal anti-phosphotyrosine antibody. RESULTS The incubation of spermatozoa in the presence of increasing concentrations of conditioned medium (CM) proteins caused a dose-dependent decrease in both tyrosine phosphorylation of sperm proteins and in the level of AR induction. When CM was removed from the sperm incubation media, the effects were reversed. Heat-inactivated CM did not affect either tyrosine phosphorylation or the induction of AR. CONCLUSIONS The present data suggest that proteins secreted from human oviductal tissue are able to inhibit events associated with sperm capacitation in vitro.

A protein isolated from human oviductal tissue in vitro secretion, identified as human lactoferrin, interacts with spermatozoa and oocytes and modulates gamete interaction

STUDY QUESTION Is lactoferrin (LF) (detected in oviductal secretion) able to bind to oocytes and sperm and modulate gamete interaction? SUMMARY ANSWER LF binds to zona pellucida (ZP) and spermatozoa (depending upon the capacitation stage and acrosome status) and inhibits gamete interaction in vitro. WHAT IS KNOWN ALREADY Proteins from human oviductal tissue secretion modulate gamete interaction and parameters of sperm function in vitro and some of them bind to sperm, but they remain to be isolated and identified. STUDY DESIGN, SIZE, DURATION Proteins were isolated from human oviductal tissue secretion using their sperm membrane binding ability. One of the isolated proteins was identified as human LF and immunolocalized in tubal tissues. LF expression was analyzed in native oviductal fluid and oviduct epithelial cells (at different phases of the menstrual cycle: proliferative, periovulatory and secretory). In addition, the LF binding sites on spermatozoa (at different capacitation and acrosome reaction stages) and on ZP and the dose-dependent effect of LF on gamete interaction were investigated. All experiments were performed at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS Tubal tissues obtained from premenopausal patients (scheduled for hysterectomy, n = 23) were cultured in DMEM/Hams F12 medium and conditioned media (CM) were collected. Motile spermatozoa were obtained by swim-up from normozoospermic semen samples from healthy donors (n = 4). An affinity chromatography with sperm membrane extracts was used to isolate proteins from CM. Isolated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophresis and further identified by nano liquid chromatography tandem mass spectrometry peptide sequencing. The presence of LF in oviductal tissue was investigated by immunohistochemistry and immunofluorescence and was detected in native oviductal fluid and oviduct epithelial cells homogenates by western blot. LF binding sites on gametes were investigated by incubating gametes with the protein coupled to fluorescein isothiocyanate (FITC). The acrosome reaction was assessed with Pisum sativum agglutinin conjugated with rhodamine. The effect of increasing concentrations of LF (0.1-100 µg/ml) on gamete interaction was evaluated by a sperm-ZP binding assay, using human oocytes donated by women undergoing IVF procedures. MAIN RESULTS AND THE ROLE OF CHANCE A protein isolated by the affinity column was identified as human LF. LF was immunolocalized in human oviductal tissue and detected in oviductal fluid and oviduct epithelial cell homogenates. In the latter case, LF expression was highest at the periovulatory phase of the menstrual cycle (P < 0.01). Different LF binding patterns were observed on spermatozoa depending upon capacitation stage and if the acrosome reaction had occurred. Unstained sperm were most prevalent before capacitation, but after incubation for 6 h under capacitating conditions and in acrosome-reacted sperm LF binding was observed, mainly localized in the equatorial segment and post-acrosomal region of the sperm head. LF binding studies on ZP showed homogenous staining. LF caused a dose-dependent significant inhibition of sperm-ZP interaction, and the effect was already significant (P < 0.01) with the lowest LF concentration used. LIMITATIONS, REASONS FOR CAUTION This study has investigated the effect of LF only on human gamete interaction in vitro and thus has some limitations. Further investigations of the potential mechanisms involved in LF action both on gamete function in vitro and in vivo in animal models are needed to confirm the role of this protein in the reproductive process. WIDER IMPLICATIONS OF THE FINDINGS The present data indicate that human oviductal LF expression is cycle dependent and inhibited gamete interaction in vitro. No previous data were available about potential direct effects of LF on gamete interaction. It could be thought that the protein is involved in the regulation of the reproductive process, perhaps contributing to prevent polyspermy. Thus, further research is needed to clarify the potential role of LF in the regulation of the fertilization process. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from FONCYT (PICT 01095, S.A.G., M.J.M) and SECyT UNR (PIDBIO238, S.A.G). The authors have no conflict of interest to declare.

Detection of mammaglogin A in blood from breast cancer patients, before and after treatment, using a one-tube nested PCR protocol. Association with the absence of tumor estrogen receptors.

OBJECTIVE A one-tube nested RT-PCR protocol was set up and used to detect mammaglobin A (MGA) expression in blood samples from breast cancer patients. The correlation of MGA detection with prognostic factors was analyzed. DESIGN AND METHODS Total RNA from nucleated blood cells was extracted from 65 breast cancer patients (before surgery and after the treatments) and 18 healthy subjects and used to detect MGA expression by a modified nested RT-PCR. RESULTS MGA expression was detected in 38.4% of patients before surgery, and in 50% and 36.8% of post-treatment samples from patients that expressed MGA or were MGA negative before surgery, respectively. MGA detection was associated with the absence of tumor estrogen receptors (p=0.004). CONCLUSIONS MGA detection by the modified nested RT-PCR is a specific marker for circulating tumor cells in patients with breast carcinoma and a negative prognostic factor for the disease.

Effects of ulipristal acetate on sperm DNA fragmentation during in vitro incubation

Abstract Objective Ulipristal acetate (UPA) acts as an emergency contraceptive by inhibiting ovulation. This study explores possible additional effects on the fragmentation of sperm DNA during in vitro incubation. Methods Motile spermatozoa from healthy donors were selected by swim-up and incubated under capacitating conditions in control medium or with UPA (1, 10, 100, 1,000 or 10,000 ng/ml). In some experiments, 200 μM of H2O2 were added to induce oxidative stress. The sperm chromatin dispersion test was performed to analyse DNA integrity (400 cells; 1000×). Lipid peroxidation (thiobarbituric acid assay), induced-acrosome reaction (AR) and sperm vitality (Eosin Y) were also evaluated in spermatozoa exposed to UPA and/or H2O2. Results During sperm incubation, the percentage of fragmented DNA increased significantly, from 15.0 ± 1.3 to 41.0 ± 4.5% (p < 0.001). In the presence of UPA, DNA fragmentation decreased significantly (p < 0.05), in a dose-dependent manner. At 100 and 1000 ng/ml, UPA also counteracted the effect of H2O2 and prevented DNA fragmentation. No effect on sperm vitality, lipid peroxidation or induced-AR was found with any treatment. Conclusions During in vitro sperm capacitation DNA fragmentation increased but the latter was counteracted in the presence of UPA, which possibly acted as a scavenger of reactive oxygen species produced by spermatozoa.

In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation

The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham′s F10 medium plus bovine albumin at 37° and 5% CO2 for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen‐free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.

Study of the effect of ulipristal acetate on human sperm ability to interact with tubal tissue and cumulus-oocyte-complexes☆☆☆

OBJECTIVE Ulispristal acetate (UPA) is a selective progesterone receptor modulator widely used for emergency contraception (EC). The described main mechanism of action is by inhibiting or delaying ovulation; however, the postovulatory effects of the drug are still on debate. Therefore, the aim of this study was to determine whether UPA could interfere with human sperm fertilizing ability. STUDY DESIGN Human motile spermatozoa were incubated under capacitating conditions with or without UPA, and then used to inseminate human tubal explants, mouse cumulus-oocyte complexes and zona-free hamster eggs. The ability of UPA to interact with human sperm progesterone (P)-binding sites was investigated by incubating the cells with fluorescent-labeled P and analyzing them by fluorescence microscopy. RESULTS UPA did not affect the ability of human sperm to bind to human tubal tissue explants surface or to penetrate the mouse cumulus mass and the zona-free hamster eggs. In addition, concentrations of UPA much higher than those present in the plasma of EC pill users were required to bind to human sperm P-binding sites. CONCLUSIONS Our study supports a lack of an agonist or antagonist action of UPA on different functional parameters associated with the fertilizing ability of human sperm. IMPLICATIONS This study provides new functional evidence supporting that the contraceptive action of UPA is not related to effects on human sperm cells, contributing to a better understanding of the mechanism of action of UPA as EC.

Use of Assisted Reproduction Techniques in Male Pathologies: Molecule Based Sperm Selection Methods

Infertility is a widespread problem that is found in over 15% of couples. In 50 % of the cases, the male factor is the key. In order to improve pregnancy rates in assisted reproductive techniques (ART), new methods to select sperm subpopulations are required. These methods are under constant evaluation, and their possible genetic and epigenetic effects on the offspring must be considered. New concepts such as DNA fragmentation and sperm apoptosis are being introduced in andrology and are considered to develop new techniques. Recently, molecule-based methods have been developed for the purpose of overcoming deficiencies in classical selection methods that involve sperm morphology and movement. One of these methods is physiologic intracytoplasmic sperm injection (PICSI), which selects spermatozoa through their property to bind to hyaluronic acid, a component of the cell-oocyte complexes that acts as a natural selector of spermatozoa. But the top rated method is based on the property of the protein annex in A5 (ANXA V) to bind to phosphatidylserine (PS), a marker of apoptosis. Magnetic-Activated Cell Sorting (MACS) conjugated with ANXA V (MACS-ANXA V) is used to remove apoptotic sperm with exposed PS from a semen sample. The use of MACS-ANX V seems to produce good quality sperm when it is evaluated by in vitro assays. But when MACS-ANXA V is used previous to ART, controversies persist regarding the benefits of sperm selection in reproductive outcomes. In this review, we consider the use of MACS-ANXA V, particularly in cases of male pathologies.