Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.     

Impact of Cu and Fe concentrations on oxidative damage in male infertility

Oxidative stress in the reproductive system is thought to have an effect on the fertilizing ability of sperm. The purpose of this study was to assess the interaction of iron (Fe) and copper (Cu) ions in suspected subfertile and fertile male groups and to find out the relationships of the semen parameters (sperm count, motility, and abnormal morphology), glutathione, malondialdehyde, and reactive oxygen species with these variables. Semen and blood obtained from 60 subfertile men and from 40 fertile volunteers were examined. The sperm count and motility in the subfertile male group were found lower than those in fertile male group (p<0.001). Cu levels in serum and seminal plasma in the subfertile male group were significantly higher than those in the fertile male group (p<0.001 and p<0.05, respectively). There was also a significant increase in the Fe level of seminal plasma in the subfertile male group (p<0.001). However, there was no significant difference in the Fe level of serum in the subfertile male group. In conclusion, these findings suggest that Cu and Fe might be mediators of the effects of oxidative damage and play an essential role in spermatogenesis and male infertility; the determination of Fe and Cu levels in serum and seminal plasma during infertility investigation is recommended.     

Making Technology Familiar: Orthodox Jews and Infertility SUPPORT, Advice, and Inspiration

This paper examines how orthodox Jews use traditional strategies and new media simultaneously to cope with infertility in the age of new reproductive technologies. Not only have they used the Internet to establish support, information, and educational networks, but also they have created frameworks for unique professional collaborations among rabbis, doctors, and clinic personnel in order to ensure that their fertility treatments are conducted with strict attention to Jewish legal concerns, particularly with regard to incest, adultery, and traditional practices regarding bodily emissions. Throughout these processes, they have innovated a hybrid language for describing and explaining infertility treatments that blends Hebrew prayers, Yiddish aphorisms, English slang, Gematria (numerology), and biomedical terminology. By using idiomatic language and folk practice, orthodox Jews construct a unique terrain that shapes and makes familiar their experience and understanding of fertility treatment. Biomedicine in this context is understood as a set of tools and strategies that can be readily appropriated and harnessed to a particular set of individual and collective goals.     

Increased luminal pH in the epididymis of infertile c-ros knockout mice and the expression of sodium-hydrogen exchangers and vacuolar proton pump H+-ATPase

Transgenic mice targeted for the c-ros gene, which are fertile when heterozygous (HET), but infertile when homozygous (knockout, KO) and associated with failure in pubertal differentiation of the epididymal initial segment, provide a model for studying the role of the epididymal luminal environment in sperm development. Luminal fluid from the cauda epididymidis was measured by both ion-selective microelectrodes and pH strips to be 0.3 pH units higher in the KO than HET. Of the genes responsible for luminal acidification, expression of mRNA of vacuolar H(+)-ATPase was found in all epididymal regions, but with no difference between KO and HET. Immunohistochemistry showed its presence in epithelial apical cells and clear cells. The Na(+)-hydrogen exchanger NHE2 was expressed at mRNA and protein levels in the caput but only marginally detectable if at all in the distal epididymis. This was compensated for by NHE3 which was expressed strongest in the cauda region, in agreement with immunohistochemical staining. Quantification of Western blot data revealed slight, but significant, decreases of NHE2 in the caput and of NHE3 in the cauda in the KO mice. The increase in luminal fluid pH in the KO mice could also be contributed to by other epithelial regulating factors including the Na(+)-dependent glutamate transporter EAAC1 formerly reported to be down regulated in the KO.     

Motor apparatus in human spermatozoa that lack central pair microtubules

Electron microscopic examination of the spermatozoa from a man suffering from asthenozoospermia (poor or low sperm motility) showed that approximately 92% of the sperm flagella lacked central pair microtubules but possessed dynein arms and radial spokes while a small percentage of the spermatozoa had complete flagella. The characteristics of the motor apparatus of the spermatozoa and the effects of caffeine on the sperm motility were examined, as were the reactivation of demembranated spermatozoa and the sliding of doublet microtubules. Almost all spermatozoa were immotile in a Tyrode solution while only a small percentage of spermatozoa showed slow forward movement or feeble flagellar vibration, whereas addition of caffeine to the sperm suspension induced forward swimming of approximately half of the spermatozoa. The reactivation of demembranated spermatozoa with MgATP(2-) could not succeed because of disintegration of the demembranated flagella. However, when the demembranated spermatozoa were exposed to MgATP(2-) and then treated with elastase, the microtubular doublets of approximately half the number of the flagella slid from the end or middle of the flagella. These results suggest that the motor apparatus in the sperm flagella that lack the central pair microtubules is functionally assembled and intrinsically capable of undergoing flagellar movement but not strong enough to beat normally.     

Leptin and metabolic control of reproduction

Leptin treatment prevents the effects of fasting on reproductive processes in a variety of species. The mechanisms that underlie these effects have not been elucidated. Progress in this area of research might be facilitated by viewing reproductive processes in relation to mechanisms that maintain fuel homeostasis. Reproduction, food intake, and fuel partitioning can be viewed as homeostatic responses controlled by a sensory system that monitors metabolic signals. These signals are generated by changes in intracellular metabolic fuel availability and oxidation rather than by changes in the amount of body fat or by changes in any aspect of body composition. Leptin might be viewed as either a mediator or as a modulator of the intracellular metabolic signal. Consistent with its purported action as a mediator of the metabolic signal, leptin synthesis and secretion are influenced acutely by changes in metabolic fuel availability, and these changes might lead to changes in reproductive function. The effects of leptin treatment on reproduction are blocked by treatments that inhibit intracellular fuel oxidation. Metabolic signals that inhibit reproduction in leptin-treated animals might act via neural pathways that are independent of leptin's action. Alternatively, both leptin and metabolic inhibitors might interact at the level of intracellular fuel oxidation. In keeping with the possibility that leptin modulates the metabolic signal, leptin treatment increases fuel availability, uptake, and oxidation in particular tissues. Leptin might affect reproduction indirectly by altering fuel oxidation or other peripheral processes such as gastric emptying. Reproductive processes are among the most energetically expensive in the female repertoire. Because leptin increases energy expenditure while simultaneously inhibiting energy intake, it may have limited use as a long-term treatment for infertility.     

Clinical implications of sperm DNA damage in IVF and ICSI: updated systematic review and meta-analysis

The clinical effect of sperm DNA damage in assisted reproduction has been a controversial topic during recent decades, leading to a variety of clinical practice recommendations. While the latest European Society of Human Reproduction and Embryology (ESHRE) position report concluded that DNA damage negatively affects assisted reproduction outcomes, the Practice Committee of the American Society for Reproductive Medicine (ASRM) does not recommend the routine testing of DNA damage for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Herein, our aim was to perform a systematic review and meta-analysis of studies investigating whether sperm DNA damage affects clinical outcomes in IVF and ICSI, in order to contribute objectively to a consistent clinical recommendation. A comprehensive systematic search was conducted according to PRISMA guidelines from the earliest available online indexing year until March 2020, using the MEDLINE-PubMed and EMBASE databases. We included studies analysing IVF and/or ICSI treatments performed in infertile couples in which sperm DNA damage was well defined and assessed. Studies also had to include information about pregnancy, implantation or live birth rates as primary outcomes. The NHLBI-NIH quality assessment tool was used to assess the quality of each study. Meta-analyses were conducted using the Mantel–Haenszel method with random-effects models to evaluate the Risk Ratio (RR) between high-DNA-damage and control groups, taking into account the 95% confidence intervals. Heterogeneity among studies was evaluated using the I² statistic. We also conducted sensitivity analyses and post-hoc subgroup analyses according to different DNA fragmentation assessment techniques. We identified 78 articles that met our inclusion and quality criteria and were included in the qualitative analysis, representing a total of 25639 IVF/ICSI cycles. Of these, 32 articles had sufficient data to be included in the meta-analysis, comprising 12380 IVF/ICSI cycles. Meta-analysis revealed that, considering IVF and ICSI results together, implantation rate (RR = 0.74; 95% CI = 0.61–0.91; I² = 69) and pregnancy rate (RR = 0.83; 0.73–0.94; I² = 58) are negatively influenced by sperm DNA damage, although after adjustment for publication bias the relationship for pregnancy rate was no longer significant. The results showed a non-significant but detrimental tendency (RR = 0.78; 0.58–1.06; I² = 72) on live birth rate. Meta-analysis also showed that IVF outcomes are negatively influenced by sperm DNA damage, with a statistically significant impact on implantation (RR = 0.68; 0.52–0.89; I² = 50) and pregnancy rates (RR = 0.72; 0.55–0.95; I² = 72), although the latter was no longer significant after correction for publication bias. While it did not quite meet our threshold for significance, a negative trend was also observed for live birth rate (RR = 0.48; 0.22–1.02; I² = 79). In the case of ICSI, non-significant trends were observed for implantation (RR = 0.79; 0.60–1.04; I² = 72) or pregnancy rates (RR = 0.89; 0.78–1.02; I² = 44), and live birth rate (RR = 0.92; 0.67–1.27; I² = 70). The current review provides the largest evidence to date supporting a negative association between sperm DNA damage and conventional IVF treatments, significantly reducing implantation and pregnancy rates. The routine use of sperm DNA testing is therefore justified, since it may help improve the outcomes of IVF treatments and/or allow a given couple to be advised on the most suitable treatment. Further well-designed controlled studies on a larger number of patients are required to allow us to reach more precise conclusions, especially in the case of ICSI treatments.     

Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.     

Blastocysts derivation from somatic cell fusion with premature oocytes (prematuration somatic cell fusion)

This study was undertaken to investigate the development of immature oocytes after their fusion with male somatic cells expressing red fluorescence protein (RFP). RFP‐expressing cells were fused with immature oocytes, matured in vitro and then parthenogenetically activated. Somatic nuclei showed spindle formation, 1st polar body extrusion after in vitro maturation and protruded the 2nd polar body after parthenogenetic activation. RFP was expressed in the resultant embryos; two‐cell stage and blastocysts. Chromosomal analysis showed aneuploidy in 81.82% of the resulting blastocysts while 18.18% of the resulting blastocysts were diploid. Among eight RFP‐expressing blastocysts, Xist mRNAs was detected in six while Sry mRNA was detected in only one blastocyst. We propose “prematuration somatic cell fusion” as an approach to generate embryos using somatic cells instead of spermatozoa. The current approach, if improved, would assist production of embryos for couples where the male partner is sterile, however, genetic and chromosomal analysis of the resultant embryos are required before transfer to the mothers.