Ana Paula Sousa

Not all sperm are equal: functional mitochondria characterize a subpopulation of human sperm with better fertilization potential.

Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality.

Dual use of Diff-Quik-like stains for the simultaneous evaluation of human sperm morphology and chromatin status

BACKGROUND Sperm chromatin status and nuclear DNA damage can be detected using well-established assays. However, most techniques are time-consuming and/or involve elaborate protocols and equipment. We have recently developed a simple and fast method to monitor sperm chromatin status in field conditions using the Diff-Quik assay which is employed in fertility clinics to assess sperm morphology with standard bright field microscopy. In the present study, we demonstrate that any Diff-Quik-like stain can easily, reproducibly and routinely monitor human sperm chromatin status as well. METHODS Different Diff-Quik-like stains were used to assess sperm morphology and the presence of abnormal dark nuclear staining in human sperm from four ART centres. The TUNEL assay was performed in the same samples, and fertility outcomes were assessed. RESULTS A significant correlation was found between TUNEL-positive sperm and dark sperm nuclei. Moreover, associations were also found between the percentage of dark sperm nuclei and seminal parameters, embryo development rate, embryo quality and clinical pregnancy, as well as with cryptorchidism, and there was a tendency towards an association with age. A value of 32% abnormal staining is suggested as a predictive threshold for embryo development and pregnancy. CONCLUSIONS Our results show that any Diff-Quik-like stain, already implemented in most laboratories to assess sperm morphology, can be adapted as an indicator for chromatin status in human sperm.

Exogenous glucose improves long-standing human sperm motility, viability, and mitochondrial function

A relevant fraction of human sperm can be maintained in simple phosphate-buffered saline (PBS) for more than 1 week at room temperature, a time that could be doubled if glucose was available. This does not seem to be directly due to sperm mitochondrial activity.

Evaluation of human sperm chromatin status after selection using a modified Diff-Quik stain indicates embryo quality and pregnancy outcomes following in vitro fertilization

Sperm chromatin/DNA damage can be measured by a variety of assays. However, it has been reported that these tests may lose prognostic value in Assisted Reproductive Technology (ART) cycles when assessed in post‐prepared samples, possibly due to the normalizing effect promoted by sperm preparation procedures. We have recently implemented a modified version of the Diff‐Quik staining assay that allows for the evaluation of human sperm chromatin status in native samples, together with standard sperm morphology assessment. However, the value of this parameter in terms of predicting in vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) outcomes after sperm selection is unknown. In this study, data from 138 couples undergoing in vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) treatments showed that sperm chromatin integrity was significantly improved after density gradient centrifugation and swim up (p < 0.001), but no correlations were found with fertilization or embryo development rates (p > 0.05). However, sperm samples presenting lower percentages of damaged chromatin were associated with better quality (Grade I) embryos in both ART procedures (p < 0.05) and clinical pregnancy among IVF couples (p < 0.05). Furthermore, regression analysis confirmed the clinical value of Diff‐Quik staining in predicting IVF (but not ICSI) clinical pregnancy (OR: 0.927, 95% CI: 0.871–0.985, p = 0.015), and a threshold value of 34.25% for this parameter was established. The proportion of IVF couples achieving a clinical pregnancy was reduced 1.9‐fold when the percentage of abnormal dark staining was ≥34.25% (p = 0.05). In conclusion, the Diff‐Quik staining assay provides useful information regarding ART success, particularly in IVF cycles, where some degree of ‘natural’ sperm selection may occur; but not in ICSI, where sperm selection is operator dependent. This quick and low‐cost assay is suggested as an alternative method to detect sperm chromatin status in minimal clinical settings, when no other well‐established and robust assays (e.g. Sperm chromatin structure assay, terminal deoxynucleotidyl transferase‐mediated dUDP nick‐end labelling) are available.

Low amounts of mitochondrial reactive oxygen species define human sperm quality.

We have applied the mitochondria-specific superoxide fluorescent probe MitoSOX Red (MitoSOX) to detect mitochondria-specific reactive oxygen species (mROS) production in human sperm samples using flow cytometry. We show that human ejaculates are heterogeneous in terms of mROS production, with three subpopulations clearly detectable, comprising sperm that produce increasing amounts of mROS (MitoSOX-, MitoSOX+, and MitoSOX++). The sperm subpopulation producing the lowest amount of mROS represented the most functional subset of male gametes within the ejaculate, as it was correlated with the highest amount of live and non-apoptotic sperm and increased both in samples with better semen parameters and in samples processed by both density-gradient centrifugation and swim-up, both known to select for higher quality sperm. Importantly, the MitoSOX- subpopulation was clearly more prevalent in samples that gave rise to pregnancies following assisted reproduction. Our work, therefore, not only describe discreet human sperm heterogeneity at the mROS level but also suggests that mROS may represent a strategy to both evaluate sperm samples and isolate the most functional gametes for assisted reproduction.

LOCALIZATION OF SNARES, NSF AND CAVEOLIN 1 IN HUMAN SPERMATOZOA: RELATIONSHIP WITH SEMINAL PARAMETERS

Membrane fusion is a very important process in gametes. The mechanism of membrane fusion during the AR has been proposed to involve SNAREs. Our aim is to quantify patterns of localization of Caveolin 1, SNAREs (Syntaxin 1A, Syntaxin 2 and VAMP 1) and NSF on human sperm, to determine how the differential distribution of these proteins might be interdependent and to evaluate if this distribution is related with seminal parameters. These proteins are present in different regions of the head of human sperm: anterior, equatorial and posterior regions and that Syntaxin 2 and Syntaxin 1A had a slightly different pattern of labelling. The presence and localization of SNAREs, NSF and Caveolin 1 do not correlate with seminal parameters. There is significant correlation between NSF and SNAREs, which may indicate a cooperation of these proteins in membrane fusion mechanisms of human sperm.

Anterior positioning of sex chromosomes on the head of human sperm sorted using visible wavelengths.

The human ejaculate contains subpopulations of sperm with distinct properties. Human X- and Y-bearing sperm were separated with fluorescence activated cell sorting. To avoid the use of UV light the quantitative DNA dyes DRAQ5® and Dyecycle™ Vybrant® Violet were used. Sorting efficiency was similar for both dyes, but lower than what is usually obtained with the classical method involving Hoechst 33342 and UV light (60-70% enrichment, versus 80-90%). A total of 2,739 spermatozoa were evaluated, from seven distinct samples using fluorescence in situ hybridization (FISH) chromosomal probes. No differences were found in sorted and unsorted populations in terms of chromosome positioning, and numeric chromosomal anomalies were not more evident following cell sorting. Furthermore in both sorted and unsorted populations the sex chromosomes were clearly located in the anterior portion of the sperm head, while a control autosome (chromosome 18) showed no such tendency, confirming previous findings. These results suggest that other quantitative DNA dyes may be used for sex chromosome-based human sperm sorting, but with lower efficiency than the standard UV-Hoechst based assay.

Teaching about citric acid cycle using plant mitochondrial preparations: Some assays for use in laboratory courses*

Potato tubers and turnip roots were used to prepare purified mitochondria for laboratory practical work in the teaching of the citric acid cycle (TCA cycle). Plant mitochondria are particularly advantageous over the animal fractions to demonstrate the TCA cycle enzymatic steps, by using simple techniques to measure O2 consumption and transmembrane potential (ΔΨ). The several TCA cycle intermediates induce specific enzyme activities, which can be identified by respiratory parameters. Such a strategy is also used to evidence properties of the TCA cycle enzymes: ADP stimulation of isocitrate dehydrogenase and α‐ketoglutarate dehydrogenase; activation by citrate of downstream oxidation steps, e.g. succinate dehydrogenase; and regulation of the activity of isocitrate dehydrogenase by citrate action on the citrate/isocitrate carrier. Furthermore, it has been demonstrated that, in the absence of exogenous Mg2+, isocitrate‐dependent respiration favors the alternative oxidase pathway, as judged by changes of the ADP/O elicited by the inhibitor n‐propyl galate. These are some examples of assays related with TCA cycle intermediates we can use in laboratory courses.