Association between high-fat diet feeding and male fertility in high reproductive performance mice.

The increasing worldwide prevalence of metabolic syndrome (MetS), especially in younger populations, is a risk factor for fertility disorders. However, a direct correlation of MetS with male infertility still remains unclear. In this work, we evaluated whether MetS has a negative impact on fertility of hybrid male mice with high reproductive performance. To induce a MetS-like condition, (C57BL/6xBALB/c) F1 male mice were fed a high-fat diet (HFD, 30% fat) for 19 weeks, while controls received a normal-fat diet (NFD, 6% fat). HFD-fed animals exhibited increased body weight, hypercholesterolemia, hyperglycemia and glucose intolerance. In vivo fertilisation assays performed along the treatment period showed no differences in fertilisation nor in vitro embryo development rates between groups. While testicular weight and morphology were similar in both groups, HFD-fed mice presented lighter epididymides and higher amounts of gonadal fat. Moreover, sperm count was lower in HFD-fed mice, despite normal sperm viability, morphology, motility or acrosome reaction. Finally, no differences were observed in in vitro fertilisation rates between groups. In summary, although HFD feeding altered some reproductive parameters, it did not impair male fertility in high performance breeders suggesting the possibility that a fertility impairment could be the result of the cumulative combination of environmental and/or genetic factors.

Molecular mechanisms involved in gamete interaction: evidence for the participation of cysteine-rich secretory proteins (CRISP) in sperm-egg fusion.

Epididymal protein DE and testicular protein Tpx-1 are two cysteine-rich secretory proteins also known as CRISP-1 and CRISP-2, respectively. DE/ CRISP-1 is localised on the equatorial segment of acrosome-reacted sperm and participates in rat gamete fusion through its binding to egg-complementary sites. Recent results using bacterially-expressed recombinant fragments of DE as well as synthetic peptides revealed that the ability of DE to bind to the egg surface and inhibit gamete fusion resides in a region of 12 amino acids corresponding to an evolutionary conserved motif of the CRISP family (Signature 2). Given the high degree of homology between DE/CRISP-1 and Tpx-1/CRISP-2, we also explored the potential participation of the testicular intra-acrosomal protein in gamete fusion. Results showing the ability of recombinant Tpx-1 to bind to the surface of rat eggs (evaluated by indirect immunofluorescence) and to significantly inhibit zona-free egg penetration, support the participation of this protein in gamete fusion through its interaction with egg-binding sites. Interestingly, rat Tpx-1 exhibits only two substitutions in Signature 2 when compared to this region in DE. Together, these results provide evidence for the involvement of both epididymal DE/CRISP-1 and testicular Tpx-1/CRISP-2 in gamete fusion suggesting the existence of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilisation.

Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction.

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.

Studies on the participation of epididymal sperm protein DE/CRISP-1 in egg activation.

Protein DE (32 kDa) associates with sperm during epididymal maturation and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. In the present work we investigated the participation of DE in two mechanisms probably involved in egg activation: the ability of DE to trigger activation by its interaction with the binding sites on the egg surface (receptor model) and its ability to regulate intracellular calcium channels (sperm factor model). The incubation of eggs with DE did not promote activation parameters such as calcium oscillations or meiosis resumption. Secondly, microinjection of DE into eggs was ineffective in either eliciting calcium release or modifying oscillations induced by an activating sperm extract. Together, these results argue against the participation of DE in egg activation, restricting the activity of this protein and its egg binding sites to the sperm-egg fusion process.

Molecular mechanisms involved in mammalian gamete fusion.

Fusion between gametes is a key event in the fertilization process involving the interaction of specific domains of the sperm and egg plasma membranes. During recent years, efforts have been made toward the identification of the specific molecular components involved in this event. The present work will focus on the best characterized candidates for mediating gamete membrane fusion in mammals. These molecules include members of the ADAM (a disintegrin and a metalloprotease domain) family, i.e., testicular proteins fertilin alpha, fertilin beta, and cyritestin, which are thought to interact with integrins in the egg plasma membrane through their disintegrin domains, and a member of the cysteine-rich secretory proteins (CRISP) family, i.e., epididymal protein DE, which participates in an event subsequent to sperm-egg binding and leading to fusion through specific complementary sites localized on the fusogenic area of the egg surface. The identification and characterization of these molecules will contribute not only to a better understanding of the molecular mechanisms underlying mammalian sperm-egg fusion but also to the development of new methods for both fertility regulation and diagnosis and treatment of human infertility.

Evidence that human epididymal protein ARP plays a role in gamete fusion through complementary sites on the surface of the human egg.

Human epididymal sperm protein ARP, a member of the cysteine-rich secretory proteins (CRISP) family, exhibits significant homology with rat epididymal protein DE, a candidate molecule for mediating sperm-egg fusion in rodents. The aim of this study was to investigate the involvement of ARP in human gamete fusion. Sequential extraction of proteins from ejaculated human sperm revealed the existence of a population of ARP that is tightly associated with the sperm surface and thus, potentially capable of participating in gamete interaction. Exposure of capacitated human sperm to a polyclonal antibody against recombinant ARP (anti-ARP) produced a significant and concentration-dependent inhibition in the ability of human sperm to penetrate zona-free hamster eggs. This inhibition was not due to a deleterious effect on the gametes because anti-ARP affected neither sperm viability or motility, nor egg penetrability. The antibody did not inhibit the occurrence of spontaneous or Ca(2+) ionophore-induced acrosome reaction, nor did it inhibit the ability of sperm to bind to the oolema, supporting a specific inhibition of the antibody at the sperm-egg fusion level. As a relevant evidence for a role of ARP in gamete fusion, the existence of complementary sites for this protein on the surface of human eggs was investigated. Experiments in which zona-free human oocytes discarded from in vitro fertilization programs were exposed to ARP, fixed, and subjected to indirect immunofluorescence revealed the presence of specific ARP-binding sites on the entire surface of the human egg, in agreement with the fusogenic properties of the human oolema. Together, these results strongly support the participation of ARP in the sperm-egg fusion process, suggesting that this protein would be the functional homologue of DE in humans.

Mammalian sperm-egg fusion: evidence that epididymal protein DE plays a role in mouse gamete fusion.

Rat epididymal protein DE associates with the sperm surface during epididymal maturation and is a candidate molecule for mediating gamete membrane fusion in the rat. Here, we provide evidence supporting a role for DE in mouse sperm-egg fusion. Western blot studies indicated that the antibody against rat protein DE can recognize the mouse homologue in both epididymal tissue and sperm extracts. Indirect immunofluorescence studies using this antibody localized the protein on the dorsal region of the acrosome. Experiments in which zona-free mouse eggs were coincubated with mouse capacitated sperm in the presence of DE showed a significant and concentration-dependent inhibition in the percentage of penetrated eggs, with no effect on either the percentage of oocytes with bound sperm or the number of sperm bound per egg. Immunofluorescence experiments revealed specific DE-binding sites on the fusogenic region of mouse eggs. Because mouse sperm can penetrate zona-free rat eggs, the participation of DE in this interaction was also investigated. The presence of the protein during gamete coincubation produced a significant reduction in the percentage of penetrated eggs, without affecting the binding of sperm to the oolemma. These observations support the involvement of DE in an event subsequent to sperm-egg binding and leading to fusion in both homologous (mouse-mouse) and heterologous (mouse-rat) sperm-egg interaction. The lack of disintegrin domains in DE indicates that the protein interacts with its egg-binding sites through a novel mechanism that does not involve the reported disintegrin-integrin interaction.

Relationship between the association of rat epididymal protein "DE" with spermatozoa and the behavior and function of the protein.

Rat epididymal glycoprotein DE associates with the dorsal region of the sperm head during sperm maturation, migrates to the equatorial segment (ES) with the acrosome reaction (AR), and is involved in gamete membrane fusion. In the present study we examined the association of DE with the sperm surface and the relationship of this interaction with the behavior and function of the protein. Cloning and sequencing of DE revealed a lack of hydrophobic domains and the presence of 16 cysteine residues in the molecule. Experiments in which cauda epididymal sperm were subjected to different extraction procedures indicated that while most of the protein is removable from sperm by mild ionic strength, a low amount of DE, resistant to even 2 M NaCl, can be completely extracted by agents that remove integral proteins. However, the lack of hydrophobic domains in the molecule and the failure of DE to interact with liposomes, does not support a direct insertion of the protein into the lipid bilayer. These results, and the complete extraction of the tightly bound protein by dithiothreitol, suggest that this population would correspond to a peripheral protein bound to a membrane component by strong noncovalent interactions that involve disulfide bonds. While ELISA experiments showed that no protein could be extracted by NaCl from capacitated sperm, indirect immunofluorescence studies revealed the ability of the NaCl-resistant protein to migrate to the ES. Together, these results support the existence of two populations of DE: a major, loosely bound population that is released during capacitation, and a minor strongly bound population that remains after capacitation, migrates to the ES with the AR, and thus would correspond to the one with a role in gamete fusion.

Potential contraceptive use of epididymal proteins: immunization of male rats with epididymal protein DE inhibits sperm fusion ability.

Rat epididymal protein DE associates with the sperm surface during maturation and participates in sperm-egg fusion. Immunization of male rats with DE raised specific antibodies and produced a significant reduction in the animals' fertility. The present study focused on determining the in vivo mechanism involved in fertility inhibition. Wistar males were injected with DE, and antibody levels and animal fertility were evaluated. Results revealed an association between the two parameters, since animals with absorbance values lower than 0.5 in ELISA presented high fertility rates (66%, 100%) while those with absorbance values higher than 0.5 exhibited the lowest fertility rates (0%, 33%). Histological studies showed no evidence of orchitis, epididymitis, or vasitis in DE-immunized animals. ELISA results revealed the presence of anti-DE antibodies in epididymal and vas deferential fluids. Indirect immunofluorescence and ELISA experiments indicated that these antibodies would not interfere with the synthesis or secretion of DE or with its association with the sperm surface. Finally, while epididymal sperm recovered from DE-immunized animals presented no changes in motility, viability, or ability to undergo capacitation and acrosome reaction, they exhibited a significant decrease in their ability to fuse with zona-free eggs, with no effect on their ability to bind to the oolemma. Together these results indicate that immunization of male rats with epididymal protein DE specifically interferes with the sperm fertilizing ability, supporting the use of epididymal proteins for contraceptive vaccine development.

Mammalian sperm-egg fusion: the development of rat oolemma fusibility during oogenesis involves the appearance of binding sites for sperm protein "DE".

Rat epididymal protein DE mediates gamete fusion through complementary sites localized on the egg surface. To investigate whether these egg components are involved in the development of rat oolemma fusibility, both the presence of DE-binding components and the ability of the oolemma to fuse with sperm during oogenesis were examined. Localization of DE-complementary sites by indirect immunofluorescence revealed the absence of fluorescent labeling on growing oocytes with a diameter < 50 microns, and the presence of a uniform staining over the entire surface of germinal vesicle oocytes with a diameter > 50 microns. This localization of oolemma components changed progressively to a patchy distribution during maturation. Whereas sperm incorporation was observed only in maturing oocytes, the development of the Hoechst transfer technique to evaluate membrane fusion revealed that germinal vesicle oocytes with a diameter > 50 microns were already competent to fuse with sperm. The involvement of the DE-complementary sites in the oolemma fusibility of these oocytes was confirmed by the fact that the presence of DE during gamete coincubation significantly (p < 0.001) reduced the percentage of oocytes with fused sperm. Together, these observations indicate that the acquisition of fusibility by the rat oolemma occurs during the growth period and involves the appearance of DE-binding components on the oocyte surface. This study provides novel information on the molecular mechanism by which the mammalian egg plasma membrane becomes competent to fuse with sperm during oogenesis.

Mammalian sperm-egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion.

Rat epididymal protein DE is localized on the fusogenic region of the acrosome-reacted spermatozoa and has a potential role in sperm-egg fusion. We investigated the presence of DE binding sites on the egg surface by co-incubating zona-free eggs and capacitated sperm in different concentrations of pure DE. Results indicate that DE produced a concentration-dependent decrease in egg penetration by sperm (fusion), with almost complete inhibition at 200 micrograms/ml. This inhibition was not due to an effect of DE on initial sperm binding to the egg membrane, since the presence of this protein did not affect the percentage of oocytes with bound sperm nor the number of bound sperm per egg. Those sperm that failed to penetrate the egg in the presence of DE became able to do so after transfer of the eggs to protein- and sperm-free medium, indicating a role for DE in an event subsequent to binding and leading to fusion. Indirect immunofluorescence using a polyclonal antibody against DE revealed a patchy labeling over the entire egg surface, with the exception of the area overlying the second metaphase spindle. This conclusion was supported by the disappearance of the DE-negative area on the fertilized egg. Zona-free eggs, incubated with DE at 4 degrees C or fixed before exposure to DE, displayed a uniform staining, suggesting that the patchy labeling resulted from aggregation of DE binding sites by the purified protein. The aggregation of these egg components may represent a necessary step of the fusion process. To our knowledge, this is the first study reporting the existence and localization of complementary sites to a specific sperm protein on the plasma membrane of the mammalian egg.

Long-term consequences of Reye syndrome: a sibling-matched, controlled study of neurologic, cognitive, academic, and psychiatric function.

Sixteen survivors of Reye syndrome treated at Yale-New Haven Hospital between January, 1977, and December, 1978, received neurologic, neuromaturational, cognitive, educational, and psychiatric assessments; 12 had siblings who were also evaluated. In the RS cohort, 13 of 16 patients were in stage 3/5 coma and intracranial pressure was monitored in 12 of 16 for 4.7 days. Blood ammonia concentration was greater than 300 micrograms/ml in 13 of 16 patients with a mean peak 438. Abnormalities on neurologic examination were noted in eight RS children and in none of the siblings. No significant differences emerged on psychometric testing of RS with siblings (12 children) or on group differences (16 RS children as a group compared to 12 siblings as controls). A significant difference was noted for those four children with onset of RS under age 7 years compared to their siblings (IQ 108 vs 134). The sibling IQ-RS IQ difference was significantly correlated with age of onset of RS. Individual Full Scale IQ scores and the sibling-RS IQ differences were also correlated with severity of RS. Similar findings were observed for educational testing. Eleven of the RS children received a psychiatric diagnosis (attention deficit disorder or anxiety reaction) compared to two of the control children. Five of the RS children had experienced a significant recent life stress. As a group, children with RS remain remarkably intact; however, those with most severe RS or who were very young when affected may have some sequelae.

Behavior and learning difficulties in children of normal intelligence born to alcoholic mothers.

Children referred to the Learning Disorders Unit of the Yale-New Haven Hospital were evaluated for indications of prenatal exposure to ethanol. In a total population of 87 children, 15 were found to have a history of maternal heavy drinking during pregnancy. The 11 boys and four girls ranged in age from 6 1/2 to 18 1/2 years. Birth weights ranged from 1,580 to 3,150 gm, median weight 2,213 gm. All growth measurements were affected: head circumference 60% less than tenth percentile, height 60% less than tenth percentile, weight 74% less than twenty-fifth percentile. The children had a continuum of dysmorphic features of FAS, with an inverse relationship noted between age of presentation and intensity of dysmorphic features. All had intelligence in the average range (IQ 82 to 113), yet experienced persistent academic failure. In addition, all shared problems of activity and attention regulation. Our results suggest a continuum of teratogenic effects of ethanol on the CNS. Alcohol exposure in utero may be an important, preventable determinant of attention deficit syndromes in childhood.

CSF monoamine metabolites in children with minimal brain dysfunction: evidence for alteration of brain dopamine. A preliminary report.

Epidemiologic and pharmacologic evidence suggests that abnormalities of catecholaminergic systems in the brain play a role in the pathogenesis of minimal brain dysfunction, but previous attempts to document a neurochemical abnormality have been unsuccessful. To better define central nervous system mechanisms in children with MBD, we have utilized the probenecid loading technique to determine the concentrations of metabolites in the CSF of a clinically homogeneous group of children with MBD. CSF concentrations of homovanillic acid, the principal metabolite of dopamine, correlated directly with CSF probenecid in 26 control subjects (r = 0.05, p less than 0.01) and in six children with MBD (r = 0.91, p less than 0.05). Concentration of HVA (ng/ml) per unit of probenecid (mug/ml) was found to be significantly lower in children with MBD (9.8 +/- 1.5, mean +/- SEM) compared to those in control subjects (16.5 +/- 1.5), suggesting reduced turnover of brain dopamine in the MBD group. CSF concentrations of 5-hydroxyindoleacetic acid (5-HIAA), the principle metabolite of serotonin, did not differ significantly between the groups. Our findings indicate that there may be a neurochemical abnormality in MBD.