Germ-cell hyaluronidases: their roles in sperm function.

Hyaluronidases (hyases) are a family of enzymes that catalyse the breakdown of hyaluronic acid (HA), which is abundant in the extracellular matrix. Two unlinked gene clusters encode these six proteins: three each in the somatic (or ubiquitous) acid-active subgroup and the neutral-active germ-cell subgroup. This review analyses the data on the expression and role of hyases in gamete biology and fertilization, using electronic databases until October 2010. Evidence indicates that hyases are membrane proteins with multifunctional essential, enzymatic and non-enzymatic, roles (cumulus penetration, zona binding and HA receptor) in fertilization. While sperm adhesion molecule-1 (SPAM1), which has neutral and acidic (bimodal) activity, is the widely conserved mammalian sperm hyase, it co-exists with an acidic hyase in murine and human spermatozoa. Thus, sperm function depends on the concerted activity of both germ cell and 'somatic' hyases. Some hyases are in low abundance in the ovary, somatic testicular cells, the male accessory organs and the male and female genital tracts where they are secreted and acquired by spermatozoa. The latter opens up the possibility of treating hyase-deficient spermatozoa via assisted reproductive technology. The findings challenge the existing classification of hyases, and support the notion that hyase activities are polygenic traits controlled by as many as five hyase genes in mice. Multiple sperm hyases may function cooperatively in a quantitative system and/or serve redundant roles. Unsolved problems include functional redundancy, which can be addressed by double gene-knockouts, and identifying the murine hyase(s) involved in zona binding or whether this role shows species specificity.

An immortalized, type-1 astrocyte of mesencephalic origin source of a dopaminergic neurotrophic factor.

Rat embryonic d 14 (E14) mesencephalic cells, 2.5% of which are glioblasts, were incubated in medium containing 10% of fetal bovine serum for 12 h and subsequently expanded in a serum-free medium using basic fibroblast growth factor (bFGF) as the mitogen. On a single occasion, after more than 15 d in culture, several islets of proliferating, glial-like cells were observed in one dish. The cells, when isolated and passaged, proliferated rapidly in either a serum-free or serum-containing growth medium. Subsequent immunocytochemical analysis showed that they stained positive for GFAP and vimentin, and negative for A2B5, O4, GalC, and MAP2. Serum-free conditioned medium (CM) prepared from these cells caused a fivefold increase in survival and promoted neuritic expansion of E14 mesencephalic dopaminergic neurons in culture. These actions are similar to those exerted by CM derived from primary, mesencephalic type-1 astrocytes. The pattern of expression of the region-selective genes; wnt-1, en-1, sis showed that 70% of the cells were heteroploid, and of these, 50% were tetraploid. No apparent decline in proliferative capacity has been observed after 25 passages. The properties of this cell line, named ventral mesencephalic cell line one (VMCL1), are consistent with those of an immortalized, type-1 astrocyte. The mesencephalic origin of the cell line, and the pattern and potency of the neurotrophic activity exerted by the CM, strongly suggest that the neurotrophic factor(s) identified are novel, and will likely be strong candidates with clinical utility for the treatment of Parkinson's disease.

BrDU-Giemsa labeling studies of satellite associations in parents of children with trisomy 21 or 13.

Studies on satellite association (SA) in parents of trisomy 21 offspring have not provided meaningful comparisons of SA frequencies since the latter was not expressed as a function of cell division number. We have used BrDU-labeling to compare SA frequencies in first and second division metaphases from lymphocytes of parents with either a trisomy 21 or trisomy 13 child and a control group. Parental origin of nondisjunction was determined in three of six families using quinacrine heteromorphisms. In the two cases of trisomy 13 determined, the errors occurred in maternal meiosis. BrDU-labeled metaphases were analyzed for SA frequency in four groups: A) parents contributing the extra chromosome; B) spouses of the parents in A; C) parents (nine) in whom the origin of a trisomy 13 or 21 was unknown; and D) healthy controls (five). The mean numbers of SAs/cell and of chromosomes/SA were not significantly different among the four groups for both first and second division cells. Sex and age showed no effect on SA frequency. There were significant decreases in mean numbers of SA/cell and chromosomes/SA in second-division cells (chromatids differentially stained) compared with first-division cells (chromatids undifferentiated). In second-division cells, two-chromosome SAs of all types showed random concordant and discordant alignment in each subject. The results from this BrDU-labeling approach provide no evidence that either quantitative or qualitative parameters of SA are directly related to a tendency of nondisjunction. They also show that acrocentric nondisjunction occurs in the presence of random chromatid alignment in SAs.

Sexual behavior and Down syndrome: the biological mechanism.

Sufficient evidence of a correlation between infrequent coitus and Down syndrome (DS) has now accumulated to warrant reevaluation of the suggested biological mechanisms. The evidence provides no support for delayed fertilization as the mechanism responsible for this correlation, as was originally proposed by German [Nature 217:516-519, 1968]. A better explanation of this association is provided by the sperm aging hypothesis, which gains its support from both animal studies and chromosomal findings of a paternal contribution to DS. The animal studies supporting this hypothesis show an increased incidence of sperm-derived trisomies resulting from sperm stored for prolonged periods in the male tract. The chromosomal findings show a paternal origin in 20% of DS infants; the sperm aging hypothesis concerns the biological mechanism in this 20%. In addition to explaining the excess of DS for older mothers, the sperm aging hypothesis explains the excess for teenage unwed mothers and indicates that sperm aging from decreased ejaculatory frequency may be a cause of DS in all age groups. Testable directly in humans against German's delayed fertilization hypothesis, the sperm aging hypothesis has immediate clinical implications. It suggests 1) an approach to reduce the incidence of DS and miscarriages and 2) clinical research that will increase knowledge of the circumstances indicating a need for antenatal monitoring.

Localization of the raf-1 protooncogene on chromosome 6 of the mouse.

The murine c-raf-1 gene was localized by in situ hybridization to 6C3, proximal to c-ki-ras-2 and distal to the Igk locus. The localization of this protooncogene may be relevant to the correlation of chromosome breakpoints in neoplastic disease.

Mouse Spam1 (PH-20): evidence for its expression in the epididymis and for a new category of spermatogenic-expressed genes.

The gene for the sperm adhesion molecule 1 (PH-20), SPAM1, has been known to be testis-specific and exclusively haploid expressed. We show that in mice, the 2 common isoforms of the protein (Spam1) observed in sperm are also present in the caput, corpus, and cauda epididymides. Both qualitative and quantitative variation of expression of the protein were observed in epididymis with the highest expression detected in the corpus. The endogenous production of enzymatically active (via hyaluronidase) Spam1 by epididymal cells is supported by the detection of steady-state Spam1 epididymal messenger RNA in both wild type and germ cell-deficient mice. In situ transcript hybridization shows the transcript to be localized to the principal cells of the epithelium. The protein was similarly immunolocalized to these cells, predominantly in vesicles near the apical region. The results suggest a mechanism for transportation of Spam1 from the epididymal epithelium to sperm during their transit and storage in the cauda. None of the current categories of spermatogenic-expressed genes shows the dual transcription pattern (haploid testicular/diploid epididymal) observed for Spam1. The work also confirms and extends the finding that Spam1 is expressed in the kidney.

Analysis of the chromosome complement in outbred mouse sperm fertilizing in vitro.

The chromosome complements in a population of mouse sperm from random-bred ICR donors were analyzed at first-cleavage metaphase after in vitro fertilization (IVF) of oocytes from females of the same strain. The sperm were aged as donations occurred within an average of 31 days, either since last mating or at arrival at the animal facility in the case of virgin males. Of a total of 598 sperm complements studied from 22 sexually mature males aged 10-26 weeks old, there was one diploid complement (0.17%). The frequencies of hyperhaploidy and structural aberrations that were studied in 338 complements were 4.4% and 3.6%, respectively, giving an overall frequency of 8.0%. The hyperhaploid complements consisted of n + 1, n + 2, n + 3, and n + 7 counts, while the structural abnormalities were of the chromosome type and included large and small fragments and a possible translocation. This is the highest frequency of sperm chromosome abnormalities reported for mouse sperm obtained from males under physiological conditions and fertilized in vitro or in vivo. Sperm aging, strain, and/or technique differences are among the factors that may be responsible for this high frequency. Since the 8.0% frequency of hyperhaploidy and structural abnormalities is similar to the frequency reported for human sperm after IVF, the outbred murine in vitro fertilization system may be a useful model to study the origin of human sperm chromosome abnormalities.

Location of the 18S and 28S rRNA cistrons in the genome of the domestic rabbit (Oryctolagus cuniculus L).

The genomic location of rDNA in the rabbit was determined by in situ hybridization with (125)I-labeled ribosomal RNA (rRNA). The results demonstrate the presence of rRNA genes on chromosomes, 13, 16, and 20, all of which have secondary constrictions, and on the telomere of the long arm of 21. The number of genes, as assessed by grain counts, was not equally distributed among the chromosomes. Chromosome 20 had the most copies and along with 13 and 16 carried significantly more genes than 21.

Assignment of the rabbit genes for alpha (PHKA) and beta (PHKB) phosphorylase kinase subunits.

The chromosomal locations of the rabbit genes for the alpha and beta subunits of phosphorylase kinase (PHKA and PHKB) were determined by in situ hybridization using rabbit cDNA probes. Our results localize PHKA to the X chromosome at the proximal end of the long arm, near the centromere, and PHKB to the same location on chromosome 5. These assignments support previously reported homoeologies of rabbit and human chromosomes.

Mouse epididymal Spam1 (PH-20) is released in vivo and in vitro, and Spam1 is differentially regulated in testis and epididymis.

The sperm adhesion molecule 1 (SPAM1 or PH-20) is an important sperm surface protein with a hyaluronidase activity and bifunctional roles in mammalian fertilization. Recently we reported that in the mouse, Spam1 is synthesized independently in the testis and the epididymis, where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Here we used mouse epididymal luminal fluid and cultured epididymal epithelial cells to demonstrate that epididymal Spam1 may be a secretory protein. Using a dual environment culture chamber system in which corpus or cauda epithelial cells are cocultured with their corresponding epididymal fibroblasts in medium supplemented with androgens and epidermal growth factor, we show that in 2- to 6-day cultures Spam1 can be detected immunocytochemically in the epithelial cells. The protein was also detected by Western blot analysis in extracts of the cultured cells and in their serum-free conditioned medium, as well as in luminal fluid from fresh caput, corpus, and caudal epididymis. Importantly, it was shown to have hyaluronidase activity, using hyaluronic acid substrate gel electrophoresis, and to be expressed in greater quantities in the corpus compared with the cauda and caput. The results not only confirm our previous finding that Spam1 is synthesized in the epididymis, but extend them by showing that it is released in the luminal fluid where it may effect posttesticular maturation and function of sperm. Results from transcript analysis indicate that epididymal and testicular Spam1 are under different transcriptional regulation.

The murine Spam1 gene: RNA expression pattern and lower steady-state levels associated with the Rb(6.16) translocation.

Recently we mapped the murine Spam1 gene to the proximal region of chromosome 6 (MMU 6). Based on the map location and physiological characteristics of its encoded sperm antigen, the gene is an attractive candidate for the sperm dysfunction seen in Rb(6.16) translocation heterozygotes and the reduced fertility of homozygotes. We have analyzed the expression of Spam1 mRNA in normal and Rb(6.16) mice. The expression of Spam1 mRNA was found to be: 1) tissue specific; it is expressed exclusively in testis; and 2) developmentally regulated, with a haploid expression. Notably, the steady-state mRNA level of Spam1 in Rb(6.16) homozygotes was 25-30% of that in chromosomally normal consomic mice and those homozygous for Rb(2.8) (7.18). In Rb(6.16) and Rb(6.15) heterozygotes the levels were 61% and 66% of the normal. Studies are currently under way to determine the protein levels and gene structure of Spam1, to detect the underlying cause of the mRNA reduction associated with these translocations.

Characterization of the genomic structure of the murine Spam1 gene and its promoter: evidence for transcriptional regulation by a cAMP-responsive element.

The Sperm Adhesion Molecule 1 (SPAM1), also known as PH-20, is a sperm membrane-bound protein that has been shown to have bifunctional roles in fertilization. It is encoded by a gene that is widely conserved in mammalian species, underscoring its importance in the fertilization process. Here we determined the genomic structure of the murine homologue, Spam1, using PCR analysis, and studied its transcriptional regulation. The gene covers approximately 10.5 kb of genomic DNA, is encoded by four exons, and the splice site consensus sequence is maintained in all intron-exon junctions, similar to that reported for the human homologue. With primer extension analysis, two transcription initiation sites were detected. One was assigned to the residue C and the other (a minor site) to the residue G, at positions 1 and -56, respectively. These are at 313 and 369 nucleotides upstream of the translation initiation codon, ATG. In about 770 bp upstream region of Spam1 that has been cloned and sequenced, multiple transcription factor binding sites including a CRE (cAMP-responsive element) were found. We specifically studied the function of the eight nucleotide CRE sequence (TGATGTCA) at -57 (or -62 depending on the strain of mice) of the promoter region. It can bind to the transcription factor CREM (cAMP-responsive element modulator) in gel mobility shift assays using mouse testis nuclear extract, and the binding can be inhibited by a 28 bp oligonucleotide containing the CRE sequence. Similar binding and inhibition assays using rat nuclear extract suggest the existence of a rat CRE sequence and the involvement of CREM in rat Spam1 expression. In vitro transcription assays suggest that CRE is necessary for the transcriptional activity of the murine promoter, and Northern analysis shows that Spam1 transcripts are absent in CREM-knockout mice. The results strongly suggest that the murine Spam1 expression is under the control of CREM, and that this transcriptional regulator for Spam1 might be conserved in other mammals, at least in the rat.

Segregation products of male mice doubly heterozygous for the RB(6.16) and RB (16.17) translocations: influence of sperm karyotype on fertilizing competence under varying mating frequencies.

The meiotic segregants of male mice heterozygous for Rb(6.16)24Lub and Rb(16.17)7Bnr were viewed, for the first time, at first cleavage metaphase. Chromosomes were analyzed after G-banding, C-banding, and karyotyping. To study sperm aging effects, chromosomes of 202 one-cell zygotes derived from males mating at intervals of approximately 3, 14, and 21 days were examined. At least 89.6% of sperm-derived complements were products of 2:2 segregation; at most, a possible 6.4% were 3:1 segregants. The six expected types of 2:2 segregants, both balanced and unbalanced, were equifrequent in the total zygote population derived from sperm of all ages. When the data were analyzed according to mating frequency, the 3-day sperm population considered most likely to be fresh showed a deficiency of the segregant nullisomic for chromosome 6 and disomic for chromosome 17, when compared with the reciprocal segregant (P less than 0.025) as well as to all other 2:2 segregants (P less than 0.05). However, these sperm fertilized in greater numbers (P less than 0.01) than their reciprocal segregant (disomic for 6 and nullisomic for 17) in the 14-day sperm population. While sperm with chromosomal abnormalities are capable of fertilization, the competence of segregants nullisomic for 6 and disomic for 17 apparently depends on the prior storage period in the male. Further, the results suggest that the effect of aneuploidy on sperm function is dependent on the specific chromosome(s) involved.

Spontaneous heteroploidy in one-cell mouse embryos.

First cleavage divisions were analyzed, after chromosome banding, in 321 zygotes recovered from superovulated, outbred ICR mice 33-35 h after injection of human chorionic gonadotropin. With 72.1% of all zygotes at metaphase and late prophase analyzed, there was 87% diploidy, 3.6% trisomy, 2.5% monosomy, 0.4% structural rearrangement, and 0.93% triploidy. Aneuploid zygotes in which there were 37 or 38 chromosomes accounted for 6.5% of the population with both haploid complements analyzable. In 45 diploid zygotes in which only one complement was analyzable, there was 93.4% haploidy, 2.2% disomy, and 4.4% hypoploidy. The chromosome most often involved in aneuploidy was the 19. The parental origin of the chromosome anomalies was determined on the basis of differential condensation of the chromosomes. The source of trisomy was shown to be the male in three and the female in five of the eight cases determined. This indicates a paternal contribution to trisomy and a female nondisjunction frequency of 1.5% (five of 321). For monosomy where the origin was identified in five of the cases, the male complement was implicated in three zygotes. The single break was seen in a sperm-derived chromosome. Two of the three triploids were dispermic and one was digynic. The outbred Swiss mouse might be a useful model for studying factors responsible for the induction of cytogenetic anomalies in early development of mammals.

Second meiotic nondisjunction is not increased in postovulatory aged murine oocytes fertilized in vitro.

We used an in vitro fertilization system to examine the effects of postovulatory oocyte age on nondisjunction at the second meiotic division. After ovulatory-inducing injections of hormone, we recovered mouse oocytes either at the estimated time of ovulation (controls) or 2, 4, 5, 10, or 14 h later. Oocytes were subjected to an in vitro fertilization procedure, and chromosomal preparations were made from first cleavage metaphase eggs. The first cleavage assay reveals morphologically distinguishable paternal and maternal chromosomes. Many of the aged oocytes were activated rather than fertilized by the in vitro procedure, but could still be analyzed for nondisjunction. We found a tendency toward retention of the second polar body after 10 and 14 h aging. A total of 488 maternal genomes, 290 of which were in the control group, were analyzable for nondisjunction. Seven hyperhaploid genomes (2.4%) were observed in the controls and 6 (3.0%) in the combined aged group. The difference between these two frequencies is not significant (Gadj = 0.164, P greater than 0.50). In the aged group, one hyperhaploid genome was in the 2-h population, three in the 5-h population, and two in the 10-h population. We were unable to find any significant increase in the frequency of nondisjunction after postovulatory oocyte aging.

The murine Rb(6.16) translocation: alterations in the proportion of alternate sperm segregants effecting fertilization in vitro and in vivo.

The segregation products of the mouse Rb-(6.16)24 Lub male translocation carrier were analyzed at first cleavage metaphase to determine whether the proportion of normal, balanced, and unbalanced sperm segregants differ in fertilizations occurring in vivo and in vitro. From 34 males, the sperm genomes in 268 first-cleavage mouse embryos were analyzed cytogenetically: 137 and 131 following in vivo and in vitro fertilization, respectively. Both systems demonstrated a preponderance of alternate (67.2% and 54.2%) as compared to adjacent segregation (10.2% and 13.7% as estimated). A contingency table showed that the distribution of reciprocal alternate segregants differed significantly between the two fertilization environments (chi 2 = 20.64, P < 0.0005). Whereas chromosomally normal sperm were 3.6 times more likely than the balanced reciprocals to fertilize in vivo (78.3% normal: 21.7% balanced), 1:1 ratios were recovered following in vitro fertilization (43.7% normal: 56.3% balanced). The data also showed an excess of Y-bearing sperm with the translocation in both in vivo and in vitro fertilization groups. In the latter these segregants were 3 times more likely than X-bearing ones to effect fertilization. These data suggest a phenotypic disadvantage of translocation-X-bearing sperm, possibly mediated through altered haploid gene expression on chromosome 6 and gene expression on the Y. The results show clear evidence for prezygotic selection in vivo and indicate that the environment in which fertilization occurs significantly affects the transmission frequency of this specific translocation.

Evidence for differential maturation of reciprocal sperm segregants in the murine Rb(6.16) translocation heterozygote.

The fertilizing ability of unaged sperm and those aged experimentally in the cauda by surgically ligating the corpus epididymis in males carrying the Rb(6.16) translocation was studied. Chromosomally normal females were inseminated with unaged sperm delivered by males mating at 3-day intervals, and aged sperm were studied after matings on 6-14 postoperative days. The sperm chromosome complement was analyzed in first-cleavage metaphase zygotes after sequential G- and C-banding of the chromosomes. Of 283 metaphasic zygotes in the control group, 183 (or 64.7%) were analyzed and showed a ratio of 2.7:1 for chromosomally normal and balanced segregants of the translocation, deviating significantly (P less than 0.001) from the expected 1:1. The ratio of X- to Y-bearing sperm also deviated from expected (P less than 0.01) mostly due to a significant deficiency (P less than 0.05) of balanced sperm that were X-bearing. Fertilized oocytes were recovered from matings of 10 males on days 6-8 postoperatively, and, of 139 metaphasic one-cell zygotes, 101 (or 72.3%) were analyzed. These showed a Mendelian ratio of 1:1 for normal and balanced segregants. The sex ratio in the aged group (57Y:41X) also showed no deviation from 1:1. The results, which reveal significant physiological distortions for both the segregation and the sex ratios in males heterozygous for the Rb(6.16) translocation, suggest that differential maturation of the translocation-bearing sperm and the chromosomally normal reciprocal exists. The findings further support the concept that sperm chromosomal complement affects their maturation and function, and that factors on chromosome 6 and the X or Y chromosome additively affect sperm function.

Sperm age, sex ratio, and hyperhaploidy frequency in mice.

Physiologically aged and unaged sperm from each of 12 sexually mature B6SJLF1/J mice were used to fertilize oocytes from females of the same strain, with each male serving as its own control. Male genomes in 323 and 307 first-cleavage metaphases obtained by in vivo and in vitro fertilization, respectively, were analyzed cytogenetically, using C-banding for detection of the Y chromosome. The sex (X:Y) ratio among all zygotes resulting from in vivo fertilization was 1.18; in zygotes resulting from in vivo fertilization by aged (14-d mating intervals) sperm, however, the ratio was 1.53, which differed significantly (chi 2 = 6.72, P less than 0.01) from the theoretical value of 1.00. Comparison of the sex ratio in zygotes resulting from in vivo fertilization by unaged sperm (3-d mating intervals), 0.94, with that in zygotes resulting from fertilization by aged sperm (using a 2 x 2 contingency table) showed a significant (chi c2 = 4.19, P less than 0.05) relationship between sex ratio and sperm age. In vitro neither the combined nor the individual 3- and 14-d data deviated significantly from the expected sex ratio of 1.00. The frequency of sperm-derived hyperhaploidy did not differ significantly between the in vivo (3.4%) and in vitro (5.9%) populations, but did between unaged (2.5%) and aged (6.8%) sperm (chi c2 = 5.74, P less than 0.01). All hyperhaploid zygotes had a complement of n + 1 chromosomes, except the 14-d in vitro group, where complements of n + 2 and n + 3 chromosomes were seen. Sperm-derived polyploidy, which was observed only in the in vitro group, was independent of sperm age and occurred in 6.8% of the zygotes. These data provide support for the sperm-aging hypothesis and indicate, for the first time, an influence of sperm aging in the male genital tract on the X:Y ratio of conceptuses resulting from natural matings of chromosomally normal males.