Michael G. O’Rand

Semen parameters and fertilization of human oocytes in vitro: a multivariable analysis

Semen parameters in 195 couples undergoing in vitro fertilization and embryo transfer were studied using multivariable analysis. Semen parameters that correlated most closely with reduced ability to fertilize apparently mature oocytes were a slow rate of foreward progression of sperm and the presence of excess numbers of white cells in semen. In men with semen parameters within the normal range, the hamster egg penetration assay (HEPA) test did not add additional predictive power. In men with suspected semen abnormalities, however, a low attachment rating added some, but minimal, predictive value. None of the predictive methods reported thus far in this or other studies offers sufficient accuracy to reliably identify the men who will prove infertile for in vitro fertilization treatment.

Analysis of recombinant mouse zona pellucida protein 2 (ZP2) constructs for immunocontraception

In this study we have examined the potential of recombinant mouse zona pellucida glycoprotein 2 (ZP2) as a target for immunocontraception. Immunogenicity studies and fertility trials were performed in outbred Swiss-Webster mice using four ZP2 constructs: Val(35)-Gly(200) (ZP2(V35-G200)), Val(35)-Leu(331) (ZP2(V35-L331)), Pro(325)-Ala(637) (ZP2(P325-A637)), and Val(35)-Ala(637) (ZP2(V35-A637)). A significant antibody response occurred to three of the four immunogens, however antibodies capable of recognizing native ZP occurred only after immunization with ZP2(V35-A637) and ZP2(P325-A637). Only immunization with ZP2(V35-A637) correlated with a reduction in fertility. Examination of the physiological basis for infertility revealed that: (1) passive transfer of ZP2 antiserum induced infertility in non-immune mice; (2) ovaries of infertile mice appeared histologically normal; (3) infertile mice produced normal numbers of eggs and (4) ZP of ovulated eggs from infertile mice demonstrated a significant reduction in the number of sperm bound compared to eggs from adjuvant controls. Infertility can be caused entirely by ZP2 antibodies without the incidence of significant ovarian pathology. This study also demonstrated that immunization with the bioactive (sperm binding) region of ZP2, recombinant ZP2(V35-G200), did not result in a significant immune response that recognized native ZP or inhibited fertility. Consequently we designed a ZP2-sperm antigen construct, replacing the C-terminal region of ZP2 with Sp17. This construct proved to be immunogenic and reduce fertility while directing the immune response to the Val(35)-Gly(200) region of ZP2.

Semen amyloids participate in spermatozoa selection and clearance

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens. DOI: http://dx.doi.org/10.7554/eLife.24888.001

Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN

Semenogelin I (SEMG1) is found in human semen coagulum and on the surface of spermatozoa bound to EPPIN. The physiological significance of the SEMG1/EPPIN interaction on the surface of spermatozoa is its capacity to modulate sperm progressive motility. The present study investigates the hypothesis that the interacting surface of SEMG1 and EPPIN co-evolved within the Hominoidea time scale, as a result of adaptive pressures applied by their roles in sperm protection and reproductive fitness. Our results indicate that some amino acid residues of SEMG1 and EPPIN possess a remarkable deficiency of variation among hominoid primates. We observe a distinct residue change unique to humans within the EPPIN sequence containing a SEMG1 interacting surface, namely His92. In addition, Bayes Empirical Bayes analysis for positive selection indicates that the SEMG1 Cys239 residue underwent positive selection in humans, probably as a consequence of its role in increasing the binding affinity of these interacting proteins. We confirm the critical role of Cys239 residue for SEMG1 binding to EPPIN and inhibition of sperm motility by showing that recombinant SEMG1 mutants in which Cys239 residue was changed to glycine, aspartic acid, histidine, serine or arginine have reduced capacity to interact to EPPIN and to inhibit human sperm motility in vitro. In conclusion, our results indicate that EPPIN and SEMG1 rapidly co-evolved in primates due to their critical role in the modulation of sperm motility in the semen coagulum, providing unique insights into the molecular co-evolution of sperm surface interacting proteins.

Analysis of deoxyribonucleic acid distribution in noncleaving oocytes from patients undergoing in vitro fertilization

The purpose of this study was to describe the quantity and distribution of deoxyribonucleic acid (DNA) in oocytes that did not fertilize or did fertilize and failed to cleave, from patients who underwent in vitro fertilization. Patients were selected with at least one cleaving egg, so that the sperm population was known to be fertile, and failure of fertilization or cleavage in the remaining oocytes could be attributed to nonspermatozoan factors. The noncleaving oocytes were classified into five categories, the majority of which (71%) lacked a polar body and any morphologically identifiable nucleus or germinal vesicle. Three general defects were found: failure to replicate the DNA properly; failure to package the DNA properly; and failure to organize the nuclear material properly after sperm penetration. It is concluded that either altered stimulation protocols or altered in vitro maturation conditions are needed to increase the average number of normal embryos available for transfer.

Characterization of the Testicular Histone-Binding Protein, NASP

The nuclear autoantigenic sperm protein (NASP) was first recognized because of its autoantigenicity in males and immunological cross-reactivity with the sperm-specific autoantigen RSA (1). NASP was initially described as a highly immunogenic testis and sperm-specific protein, which was present in the postacrosomal region of mature spermatozoa and in the nucleus of developing spermatogenic cells (1,2). From DNA sequence comparisons, NASP appears to have evolved from the N1/N2 gene expressed in oocytes of Xenopus laevis (3,4). Indeed, the 3’ untranslated sequence identity between the rabbit and Xenopus mRNAs reaches approximately 60%, implying that mammalian NASP is a true homologue of N1/N2.

Fertilization in the Rabbit

Discovered by the Phoenicians in ancient times, the rabbit has been bred in captivity for hundreds of years. In the monasteries of the 16th century, domestication of the rabbit (Oryctolagus) and hare (Lepus) led to the continued breeding of rabbits because of their ability to reproduce easily in leporaria (Fox, 1974). Today’s modern laboratory rabbit is a decendent of the European rabbit Oryctolagus cuniculus and now exists in dozens of different breeds, from the 15-lb Flemish giant to the 2 1/2h-lb. pound Polish white. Because of such a long history of domestication, evolutionary changes from the true wild type have undoubtedly occurred; nevertheless, researchers have continued to study the reproductive biology of the domestic rabbit for over 300 years. In fact, early observations on ovarian follicles, which later became known as “Graafian follicles,” were made in the rabbit by De Graaf in the late 17th century. For the next 200 years numerous investigators reinvestigated ovulation and early development in the rabbit, using the rabbit as a model for mammalian development because it is easily bred in captivity and ovulation is induced by coitus. By 1839, Barry had clearly established the time of ovulation as 10 hr after coitus, and he made the significant observation that the interaction of the spermatozoon with the egg was responsible for the development of the embryo.

Inhibition of sperm motility in male macaques with EP055, a potential non-hormonal male contraceptive

Abstract Men have two practical choices for contraception; the condom which has a high typical use failure rate or vasectomy. New male hormonal and non-hormonal contraceptives are under development that target either the production of sperm (spermatogenesis) or the delivery of sperm. One particular target is the sperm protein EPPIN, which is present on the surface of human spermatozoa. EP055 is a small organic compound that targets EPPIN on the surface of sperm and inhibits motility. EP055 was tested in cynomolgus (Macaca fascicularis) males to determine its plasma half-life after intravenous (i.v.) infusion of a single dose and for binding to its target tissues. Our initial study demonstrated a plasma half-life for EP055 of 10.6 minutes. In a second study examination of macaque testis, epididymis, and plasma after i.v. infusion of a single dose of compound EP055 (63.25 mg/kg) demonstrated that EP055 was detected in testis and epididymis two hours and six hours post-infusion. We initiated a trial in rhesus (Macaca mulatta) males to assess the availability of EP055 in semen and its effect on sperm motility as a measure of the drugs efficacy. Four macaques were infused with a low dose (75–80 mg/kg) followed by a recovery period and a subsequent high dose (125–130 mg/kg) of EP055. After high dose administration, sperm motility fell to approximately 20% of pretreatment levels within 6 hours post-infusion; no normal motility was observed at 30 hours post-infusion. Recovery of sperm motility was obvious by 78 hours post-infusion; with full recovery in all animals by 18 days post-infusion. EP055 has the potential to be a male contraceptive that would provide a reversible, short-lived pharmacological alternative.