MiniBioReactor Array (MBRA) in vitro gut model: a reliable system to study microbiota-dependent response to antibiotic treatment.

Background: Antimicrobial drugs are mostly studied for their impact on emergence of bacterial antibiotic resistance, but their impact on the gut microbiota is also of tremendous interest. In vitro gut models are important tools to study such complex drug-microbiota interactions in humans. Methods: The MiniBioReactor Array (MBRA) in vitro microbiota system; a single-stage continuous flow culture model, hosted in an anaerobic chamber; was used to evaluate the impact of three concentrations of a third-generation cephalosporin (ceftriaxone) on faecal microbiota from two healthy donors (treatment versus control: three replicates per condition). We conducted 16S microbiome profiling and analysed microbial richness, diversity and taxonomic changes. ß-Lactamase activities were evaluated and correlated with the effects observed in the MBRA in vitro system. Results: The MBRA preserved each donor's specificities, and differences between the donors were maintained through time. Before treatment, all faecal cultures belonging to the same donor were comparable in composition, richness, and diversity. Treatment with ceftriaxone was associated with a decrease in α-diversity, and an increase in ß-diversity index, in a concentration-dependent manner. The maximum effect on diversity was observed after 72 h of treatment. Importantly, one donor had a stronger microbiota ß-lactamase activity that was associated with a reduced impact of ceftriaxone on microbiota composition. Conclusions: MBRA can reliably mimic the intestinal microbiota and its modifications under antibiotic selective pressure. The impact of the treatment was donor- and concentration-dependent. We hypothesize these results could be explained, at least in part, by the differences in ß-lactamase activity of the microbiota itself. Our results support the relevance and promise of the MBRA system to study drug-microbiota interactions.

Creatine kinase immunocytochemistry of human sperm-hemizona complexes: selective binding of sperm with mature creatine kinase-staining pattern.

OBJECTIVE: To examine the clinical significance of the increased sperm cytoplasmic content that is due to a fault of spermatogenesis, we have further studied the relationship between increased sperm creatine kinase (CK) concentrations and diminished fertilizing potential in men. In the present work, we used CK immunocytochemistry of human sperm-hemizona (HZ) complexes to examine whether the distribution of mature (clear heads), intermediate (sperm heads with light stippling), and immature (heads with heavy stippling or with solid CK staining) spermatozoa bound to the HZ would follow the incidence of these sperm in the samples tested, or if there is a preferential binding by the mature sperm. DESIGN: Two pairs of HZ were exposed to washed semen and to their swim-up sperm fractions. The sperm and sperm-HZ complexes were treated with a CK antibody followed by horseradish peroxidase immunostaining, and the sperm were evaluated for maturity. SETTING: Men presenting for fertility evaluation were studied in two university-based andrology laboratories. RESULTS: The binding of the HZ was selective for mature sperm as indicated by the incidence of intermediate and immature sperm in washed semen versus bound to the HZ (intermediate: 20.0% versus 1.4%; immature: 7.6% versus 0.5% [mean +/- SEM]) or in swim-up sperm fractions versus the HZ (intermediate: 18.7% versus 3.4%; immature: 2.5% versus 0.2%). The binding was almost exclusive to normal sperm (96.4% to 98.1%) whether the HZ were exposed to washed semen or swim-up fractions in spite of the five to ten times higher incidence of intermediate and immature sperm. CONCLUSIONS: Mature sperm selectively bind to the zona. We suggest that spermatozoa with immature CK-staining patterns are deficient in the site(s) of oocyte recognition and binding.

Sperm creatine phosphokinase M-isoform ratios and fertilizing potential of men: a blinded study of 84 couples treated with in vitro fertilization.

OBJECTIVE: To examine the value of sperm creatine phosphokinase M-isoform (CK-MM) measurements toward predicting fertilizing potential of men. DESIGN: In 84 in vitro fertilization (IVF) couples without knowing the semen parameters, reproductive history or the outcome of the IVF cycles, we determined the sperm CK-MM ratios (the proportion of sperm CK-MM versus CK-MM+CK-BB). Husbands with less than 10% or greater than or equal to 10% CK-MM ratios were classified as "low likelihood for fertilization" (CKMM-Infertile, n = 22) or "high likelihood for fertilization" (CKMM-Fertile, n = 62), respectively. RESULTS: Both the CKMM-Infertile and CKMM-Fertile groups (CK-MM ratios: 4.9% +/- 0.6% versus 31.1% +/- 1.8%) were in the normospermic range (31.5 +/- 6.9 versus 78.4 +/- 5.9 x 10(6) sperm/mL and 45.6% +/- 5.0% versus 54.0% +/- 2.0% motility). The fertilization rates (6.2 versus 4.9 oocytes inseminated) were 14.2% versus 53.4%, and 72.7% versus 25.8% of the couples failed to achieve any oocyte fertilization. All 14 pregnancies (16.7% rate) occurred in the CKMM-Fertile group. The pregnancy rate in the 62 CKMM-Fertile couples was 22.6%, and considering only the 46 CKMM-Fertile women in whom oocyte fertilization occurred, it was 30.4%. Among the 22 CKMM-Infertile men, 9 were normospermic and 9 of the 62 CKMM-Fertile men were oligospermic. Within the CKMM-Fertile group, 12 and 2 of the 14 pregnancies occurred by the 53 normospermic and 9 oligospermic men (22.6% versus 22.2% rate). CONCLUSIONS: Sperm CK-MM ratios, a measure of normal sperm development, predict fertilizing potential independently from sperm concentrations. Sperm CK-MM ratios also detect unexplained male infertility (infertile men with normospermic semen), a diagnosis that until now could not be substantiated.

Correlation between the rate of lipid peroxidation and cellular maturity as measured by creatine kinase activity in human spermatozoa.

We have demonstrated previously that creatine kinase (CK) activity is a measure of cellular maturity and fertilizing potential in human spermatozoa. In the present work we have examined whether there is a relationship between sperm CK activity and the rate of lipid peroxidation (LP) as measured by malondialdehyde (MDA) formation. Both MDA production and CK activity were higher in oligospermic than in normospermic specimens (P < 0.001, N = 41 and 101, respectively), and there was a close correlation (R = 0.43, P < 0.001) between these two biochemical parameters. As demonstrated previously with the CK measurements, there was a heterogeneity among the groups: About 40% of the oligospermic men had MDA and CK activity values similar to that of the normospermic group, and 12% of the normospermic men had MDA and CK activity values similar to that of the oligospermic group. We have also examined in three experimental paradigms the question of sperm-to-sperm propagation of increased LP and the possible increase in LP following centrifugation as used in sperm preparation for assisted reproduction: The MDA differences among Percoll sperm fractions originating within the same specimens, the lack of change in MDA production after co-centrifugation and co-incubation of samples with high and low sperm LP rates, and the repeated centrifugation of the same specimens without an increase in MDA production all indicated the lack of sperm-to-sperm propagation of LP or increase in LP due to mechanical stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine triphosphate (ATP) concentrations and ATP/adenosine diphosphate ratios in human sperm of normospermic, oligospermic, and asthenospermic specimens and in their swim-up fractions: lack of correlation between ATP parameters and sperm creatine kinase concentrations.

The authors had previously found an inverse correlation between per sperm creatine phosphokinase activity and sperm concentrations in men. Because creatine phosphokinase is a key enzyme in sperm energy transport, the possible relationship of sperm creatine phosphokinase activity, sperm adenosine triphosphate (ATP) concentrations, sperm ATP/ADP (adenosine diphosphate) ratios, and computer-aided semen analysis sperm motility parameters were then studied. The ATP concentrations and ATP/ADP ratios, measured by high-pressure liquid chromatography in washed sperm, were similar in normospermic and oligospermic specimens (ATP: 123.1 +/- 21.6 vs. 90.0 +/- 24.5 pmol/10(6) sperm; ATP/ADP: 2.8 +/- 0.4 vs. 2.1 +/- 0.4, N = 32 and 17, mean +/- SEM), and in samples with normal and less than 40% sperm motility (ATP: 96.8 +/- 27.2 vs. 122.2 +/- 19.6 pmol/10(6) sperm; ATP/ADP: 2.4 +/- 0.5 vs. 2.8 +/- 0.4, n = 26 and 23). In the swim-up sperm fractions, which showed improved motility, the ATP concentrations, but not the ATP/ADP ratios, were lower than in the initial semen samples (ATP: 152.9 +/- 28.4 vs. 90.3 +/- 10.6 pmol/10(6) sperm, P less than 0.05; ATP/ADP: 3.3 +/- 0.5 vs. 3.9 +/- 0.7, N = 18 pairs of samples). This is consistent with our previous finding of a lower cytoplasmic content in sperm in swim-up fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm creatine kinase activity in fertile and infertile oligospermic men.

The authors examined the value of sperm creatine-kinase (CK) activity parameters to predict sperm fertilizing potential of oligospermic men. Two patient groups from our intrauterine insemination program were studied: fertile oligospermic (32 men/46 specimens) and infertile oligospermic (19 men/82 specimens). In the initial specimens, the CK activities were (mean + SEM IU CK/10(8) sperm): 0.53 +/- 0.09 and 1.17 +/- 0.19 (P less than 0.001). The corresponding values in the swim-up fractions were 0.32 +/- 0.06 and 0.67 +/- 0.08 (P less than 0.001). In a subset of samples by fertile (N = 33) and infertile (N = 66) oligospermic men who had close to identical sperm concentrations (11.9 +/- 0.9 vs. 11.9 +/- 0.5 million sperm/ml) and motility values (23.7 +/- 1.7 vs. 23.0 +/- 1.3%), the CK activities were significantly lower in the fertile group in both the initial (P = 0.02) and in the swim-up (P = 0.002) samples. A logistic regression analysis of all 160 study samples (including 21 normal men/32 samples) further demonstrated that CK activities were predictive of fertilizing potential, whereas sperm concentrations of the samples provided no additional contribution. Sperm CK and similar biochemical markers will facilitate selection of men for various approaches in assisted reproduction.

Sperm function and choice of preparation media: comparison of Percoll and Accudenz discontinuous density gradients.

We compared the sperm populations prepared by Accudenz (35-65%) and Percoll (40-80%) density gradients in 21 normospermic specimens (concentration, 53.6 +/- 3.8 x 10(6) sperm/ml; motility, 44.5 +/- 3.5%). Accudenz facilitated a higher recovery of sperm and motile sperm (68.4 +/- 6.6% vs. 49.3 +/- 4.9%, P < 0.001, and 87.8 +/- 4.1% vs. 77.8 +/- 3.7%, P < 0.01, respectively). Sperm motility was lower in the Accudenz compared to the Percoll pellets; thus the values of total motile sperm recovered were not different (17.1 +/- 2.4 vs. 15 + /- 1 2.2 x 10(6) sperm/ml). The long term retention of sperm motility was substantially improved in Accudenz (at 24 hours, 34.9 +/- 2.8% vs. 26.3 +/- 1.5%; 60% vs. 40% of the initial motility, P < 0.001), and the Accudenz vs. Percoll samples also exhibited a higher retention of total motile sperm (at 24 hours, 9.8 +/-.2 vs. 6.1 = 0.5 x 10(6) motile sperm/ml, P < 0.05). The sperm motility index, a multiple of velocity and motility in the sample that reflects the efficiency of the sperm population in sperm-oocyte interaction, was 75% higher in the Accudenz samples at 24 hours (3.6 +/- 0.4 vs. 2.1 +/- 0.2, mu m/second, P < 0.01). Sperm cellular maturity by the creatine phosphokinase (CK) activity and CK-M to CK-B isoform ratio parameters (in the original samples 0.14 +/- 0.02 lU CK/100 x 10(6) sperm and 57.9 +/- 3.7%, respectively) were improved in both the Accudenz and Percoll pellets (P < 0.001), with no difference between the two sperm fractions. Sperm activation status monitored by chlortetracycline fluorescence indicated that after 4 hours of incubation the incidence of fully acrosome-reacted spermatozoa in the Accudenz versus Percoll pellets was 6.2 +/- 0.3% versus 13.1 +/- 1.0% (P < 0.001), a 100% increase in Percoll. We can conclude that Accudenz yields a higher concentration of motile spermatozoa, with improved retention of motility, velocity, and acrosomal integrity and without an increase of sperm with diminished cellular maturity. Thus, in sperm preparation for intrauterine insemination, in which the timing of ovulation and insemination frequently do not correspond, Accudenz-prepared sperm, with a better retention of motility/velocity and acrosomal integrity and with a consequential higher resistance to activation by the female reproductive tract, are expected to be more effective.

Cytoplasmic extrusion and the switch from creatine kinase B to M isoform are completed by the commencement of epididymal transport in human and stallion spermatozoa.

Although in several species there is a relationship between epididymal sperm transport and fertility, in human in vitro fertilization (IVF), spermatozoa recovered from the caput epididymidis or even the rete testis are fertile. We studied two objective markers of sperm maturity in the sperm of men and stallions: creatine kinase (CK) concentrations, which are a measure of cytoplasmic retention in immature spermatozoa, and the ratio of CK-M and CK-B isoforms (% CK-M/[CK-M + CK-B]), which is proportional to the incidence of mature sperm. The CK markers and the fertilizing function are closely related: Immature sperm with cytoplasmic retention do not bind to the zona, because during cytoplasmic extrusion, the sperm plasma membrane is also remodeled. We examined whether changes in sperm CK values are still ongoing during epididymal transport, or if cellular maturation is completed prior to the arrival of sperm in the caput epididymidis. The incidences of mature sperm in human caput and corpus epididymidis (studied in six men with obstructive azoospermia of various pathogeneses) were (mean+/-SEM) 55.7+/-2.2 and 49.3+/-7.6%, respectively; and the sperm CK-M ratios in the caput epididymidis of three men were 72, 75, and 70%, values that are similar to those of ejaculated sperm. In four segments of the proximal and distal epididymis of three stallions (the origin of sperm was also verified by the position of the cytoplasmic droplet) and in ejaculate of five stallions, the incidences of mature sperm were 88.2+/-6.2, 89.0+/-6.7, 90.3+/-7.8, 87.6+/-5.9, and 86.7+/-0.8%, and the respective CK-M ratios were 75.0+/-8.7, 84.2+/-2.9, 87.9+/-1.2, 92.5+/-1.5, and 69.3+/-3.5%. There were no differences in the incidences of mature and immature spermatozoa or in CK-M ratios among sperm arising from the various epididymal regions or from the ejaculate in men or stallions. Thus, the cellular maturation events in sperm, as detected by the CK markers, are completed by the time the sperm commences epididymal transport. These findings are in agreement with the IVF fertility of sperm aspirated from the male reproductive tract. The data may also suggest that the primary role of sperm epididymal transport in men is to remodel the plasma membrane to enhance sperm functional integrity in the diverse environments of the male and female reproductive tracts prior to fertilization.

Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentration and abnormal head morphology.

Our previous creatine phosphokinase (CK) activity studies in human sperm revealed differences among men and among sperm populations within the same specimen. Samples with low sperm concentrations, high incidence of abnormal sperm morphology, and diminished fertility had higher per sperm CK activity. In the present work, we demonstrated, with 14C-FDNB covalent CK active site modification and with direct CK immunocytochemistry, that the higher CK activity is related to an increased content of CK and of other proteins in sperm. Also, sperm heads with higher CK content were significantly larger and rounder and showed a higher incidence of amorph configuration. We suggest that these biochemical and morphological irregularities are related and are due to a failure of spermatogenesis, more specifically, to a higher retention of cytoplasm, which in normal sperm development is lost to the Sertoli cells as residual bodies. Thus higher CK activity and larger or irregular head size in human sperm signify cellular immaturity and a failure to complete spermatogenesis.

Spermatogenesis-related change in the synthesis of the creatine kinase B-type and M-type isoforms in human spermatozoa.

We have demonstrated earlier that the per sperm creatine-N-phosphotransferase (CK) activity was increased in oligospermic vs. normospermic men. The increased sperm CK activity is related to higher concentrations of cellular CK, which may indicate a defect of cytoplasmic extrusion during spermatogenesis. In the present work, we examined whether in spermatozoa, similar to muscle, there is a change in the synthesis of B-CK and M-CK isoforms during cellular differentiation. In 109 normospermic and 50 oligospermic specimens (sperm concentrations 60.6 +/- 3.7 vs. 8.8 +/- 1.3 million sperm/ml; all values expressed as mean +/- SEM), the relative concentrations of the M-CK isoform (M-CK/M-CK + B-CK) were 27.2% +/- 2.1% vs. 6.7% +/- 0.9% (P less than 0.001). The per sperm CK activities showed comparable differences (0.21 +/- 0.02 vs. 0.89 +/- 0.1 CK IU/100 million sperm; P less than 0.001) in the two groups, and there was a close correlation between per sperm CK activities and M-CK concentrations (R = 0.69, P less than 0.001, N = 159). This indicates that the loss of cytoplasm and the commencement of M-CK isoform synthesis are related events during the last phase of spermatogenesis, also that the incidence of spermatozoa with incomplete cellular maturation is higher in oligospermic specimens. In characterizing the M-CK, we found that sperm (unlike muscle tissue) lack the MB hybrid of CK dimers. However, in the presence of muscle M-CK, the muscle-sperm MB-CK hybrid has formed. Thus in sperm and muscle the M-CK isoforms are structurally different, whereas the B-CKs are apparently homologous.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between sperm creatine phosphokinase activity and sperm concentrations in normospermic and oligospermic men.

Toward the development of biochemical probes for the assessment of sperm function we have measured the activities of sperm creatine-N-phosphotransferase (CPK). There was a highly significant inverse correlation (P less than 0.001 in all comparisons) between sperm CPK activities and sperm concentrations in specimens of normospermic and oligospermic men with greater than 30 million sperm/ml (0.106 +/- 0.01 SEM, N = 90, expressed as CPK U/100 million sperm), 20-30 million sperm/ml (0.333 +/- 0.07 SEM, N = 30) and 10-20 million sperm/ml (0.583 +/- 0.12 SEM, N = 30) when compared with the CPK values of the less than 10 million/ml specimens (2.242 +/- 0.46 SEM, N = 30). Furthermore, the distribution of CPK activities within these four groups showed that 96%, 67%, 43%, and 4% of the samples, respectively, were in the less than 0.250 CPK U/100 million sperm normal range (mean + 2 SD of the greater than 30 million sperm/ml group). However, there was no relationship between sperm CPK activities and the values of sperm motility (P greater than 0.15) or morphology (P = 0.38) in the samples. The migrated sperm fractions (significantly improved in motility and velocity parameters) showed CPK activities lower than the initial semen specimens (P less than 0.01, N = 150). In fact, in some oligospermic men the CPK activities of the migrated sperm fractions were within the range of normospermic samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm creatine phosphokinase activity as a measure of sperm quality in normospermic, variablespermic, and oligospermic men.

We have found a significant inverse correlation between sperm concentrations and sperm creatine N-phosphotransferase (CPK) activities in oligospermic and normospermic human specimens. In the present work, we carried out serial CPK determinations to assess whether there is a relationship between fluctuating sperm concentrations and sperm quality in consistently oligospermic and variablespermic (sperm concentrations are occasionally in the greater than 20 million/ml range) husbands of 65 couples (23 normospermic men/51 samples, 25 consistently oligospermic men/80 samples, and 17 variablespermic men/68 samples). The sperm CPK activities were significantly lower in the normospermic vs. the oligospermic or variablespermic groups (p less than 0.001), but there were no differences between the latter two (p greater than 0.25). The mean CPK values of migrated sperm fractions in both the oligospermic and variablespermic populations were improved (at least 20% decline in CPK values) compared to those of the initial specimens (1.27 +/- 0.38 vs. 0.68 +/- 0.37 and 0.77 +/- 0.32 vs. 0.46 +/- 0.24 SEM U/100 million sperm, respectively, p less than 0.001 in both pairs) and the incidence of the "failed-to-improve" samples was also similar in the two groups (44/36 vs. 45/23, p greater than 0.2). The lack of differences in the mean CPK activities, in the distribution of CPK values under and over 0.250 U/100 million sperm level, and in the ratio of migrated samples with improved or with failed-to-improve CPK activities suggests that sperm quality is not different between men who are consistently oligospermic and those who occasionally produce normospermic specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin heavy chain in avian muscular dystrophy corresponds to the neonatal isozyme.

We have previously demonstrated, based on comparison of homologous amino acid sequences and of two-dimensional CNBr peptide gel patterns, that the myosin heavy chain in pectoralis muscles of Storrs, Connecticut dystrophic chickens is different from that of their normal controls (Huszar, G., Vigue, L., De-Lucia, J. Elzinga, M., and Haines, J. (1985) J. Biol. Chem. 260, 7429-7434). Others have shown, however, that genomic banks and mRNA complements of the control and dystrophic birds are not different. In the present studies, we have examined the hypothesis that the "dystrophic" myosin heavy chain is not a novel gene product, but is a developmental isozyme which is expressed in pectoralis muscles of adult chickens due to the dystrophic process. Two-dimensional maps of myosin heavy chain CNBr peptides were prepared from breast muscles of 17-day in ovo (embryonic), 25-day posthatch (neonatal), and adult birds of the Storrs dystrophic and of two control strains. Also, myosin and actomyosin ATPase enzymatic activities of the various preparations were determined in the pH range of 5.5 to 9.0. Analysis of the peptide maps demonstrates that the embyronic, neonatal, and control adult myosin heavy chain isozymes are distinctly different gene products with only minute variations between the respective developmental isozymes in dystrophic and control muscles. However, the pectoralis myosin heavy chain of adult dystrophic birds, which is a homogeneous isozyme population by amino acid sequences and gel patterns, corresponds to that of the neonatal-type myosin heavy chain. The ATPase properties of the embryonic, neonatal, or adult pectoralis myosins and actomyosins were not different, whether the level of specific activity or the pattern of pH activation is considered. Since the mobility of neonatal chicks (primarily neonatal-type isozymes) is not restricted, the differences in myosin heavy chain structures are part of the syndrome, but not the cause of avian muscular dystrophy.

Putative creatine kinase M-isoform in human sperm is identifiedas the 70-kilodalton heat shock protein HspA2.

We previously described a putative creatine kinase M isoform in human sperm that is developmentally regulated and expressed during late spermiogenesis, simultaneous with cytoplasmic extrusion. We have now identified this protein as the testis-expressed 70-kDa heat shock protein chaperone known as HspA2 (the human homologue of mouse Hsp70-2). We have isolated and characterized HspA2 (formerly CK-M) by amino acid sequencing and have localized it by immunocytochemistry to spermatocytes at low levels, to spermatids, and in the tail of mature sperm. The specificity of the CK-M/HspA2 antiserum to HspA2 was demonstrated on immunoblots of one- and two-dimensional SDS-PAGE. In agreement with our earlier biochemical data, immunocytochemistry of testicular tissue indicated that HspA2 is selectively expressed in mature spermatids and in sperm about to be released in the seminiferous tubuli. The identity of HspA2 has been further confirmed by cross-absorption of the mouse HSP70-2 antibody by the HspA2/CK-M fraction, and by identical immunostaining patterns of human testicular tissue using either the anti-CK-M/HspA2 or an anti-mouse Hsp70-2 antisera. During spermiogenesis, both cytoplasmic extrusion and plasma membrane remodeling, which facilitate the formation of the zona pellucida binding site, involve major intrasperm protein transport, which may be chaperoned by HspA2. Accordingly, in immature human sperm, which fail to express HspA2, there is cytoplasmic retention and lack of zona pellucida binding. The present findings provide the biological rationale for the role of the human HspA2 as an objective biochemical marker of sperm function and male fertility, which we have established in earlier clinical studies.

Morphometric assessment of mature and diminished-maturity human spermatozoa: sperm regions that reflect differences in maturity.

As part of our studies on sperm maturity and function, we examined the head, midpiece and tail of human spermatozoa using computerized morphometry in order to determine which regions reflect the differences between mature spermatozoa and spermatozoa of diminished cellular maturity. We studied 20 men, who were divided into two groups based on their lower (LCKM: 14.6 +/- 7.0%, n = 8) and higher sperm creatine kinase (CK-M) isoform ratios (HCKM: 48.0 +/- 4.3%, n = 12) in the initial semen. Using a sequential centrifugation method which relies on the lower density of immature spermatozoa with retained extra cytoplasm, we prepared three sperm fractions with progressively declining maturity, as confirmed with CK-M isoform ratio measurements. Following the sequential fractionation, we affixed the spermatozoa to glass slides, stained the midpiece and the sperm contour, and photographed 25 spermatozoa in each of the 60 fractions (1509 spermatozoa in all). The spermatozoa were then individually digitized on the Image-1 system, and the dimensions of the head, midpiece, and tail were determined. While the data showed significant differences in the midpiece and tail dimensions between the mature and diminished-maturity sperm fractions, the head dimensions were similar and did not reflect sperm maturity. We postulated that the relationship between the biochemical markers of sperm maturity and sperm morphology is based on common spermiogenic events. The data support this idea. In immature spermatozoa in which cytoplasmic extrusion, CK-M isoform expression, and tail sprouting are all diminished, the retained extra cytoplasm in the midpiece and shorter tail length contribute to the morphological variations that we identified by morphometry and considered in sperm morphology. These morphometric features, in association with fluorochrome-coupled biochemical probes, can facilitate the identification of mature spermatozoa in computer-assisted semen analysis.

Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios.

Sperm creatine phosphokinase (CK) concentrations and the synthesis of the CK-M isoform reflect normal spermiogenesis and predict maturity and fertilizing potential of ejaculated human spermatozoa. Immature spermatozoa, characterized by cytoplasmic retention and low CK-M to CK-B isoform ratios, are deficient in zona binding and fail to cause pregnancies. Because these sperm lack zona-binding ability, we examined in this study whether beta 1,4-galactosyltransferase (GalTase), a key element of sperm-zona interactions in mice, is diminished in immature human sperm. Unexpectedly, GalTase was overexpressed in immature sperm relative to mature sperm: the levels of cytoplasmic CK and plasma membrane GalTase were positively correlated (r = 0.78, p < 0.001, n = 88). Sperm populations with various levels of cellular maturity, prepared by Percoll gradients, had different CK and GalTase concentrations, but within each subpopulation the relationship between CK and GalTase was maintained (p < 0.01-0.001). GalTase activities in intact and vortex-disrupted sperm fractions were similar, showing that GalTase is present on the surface membrane of human sperm--similar to the situation in all other species assayed. The changes previously reported by our laboratory in zona-binding ability and lipid peroxidation rates (which occur simultaneously with cytoplasmic extrusion), decline in CK activity, and increased expression of the CK-M isoform are suggestive of a remodeling of the sperm surface concomitant with cytoplasmic maturation. The changes reported here in GalTase expression on the surface of maturing spermatozoa prove this hypothesis.

Biochemical markers of early and late spermatogenesis: relationship between the lactate dehydrogenase-X and creatine kinase-M isoform concentrations in human spermatozoa.

As part of our research program on biochemical markers of sperm maturity, we have studied sperm creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations and the isoform ratios of the CK-M [% CK-M/(CK-M + CK-B)] and LDH-X [% LDH-X/(LDH-X + LDH-a)] in 50 oligospermic and 95 normospermic men [corrected]. Because the synthesis of LDH-X is initiated in early spermatogenesis, and that of CK-M commences in late spermiogenesis simultaneously with cytoplasmic extrusion, we proposed two working hypotheses:(1) LDH and CK concentrations reflect the retained cytoplasm in sperm, thus the activities of both enzymes will be related and will be higher in oligospermic specimens, which have a higher incidence of immature spermatozoa; and (2) because in normally developed sperm both LDH-X and CK-M are present, there will be a correlation between LDH-X and CK-M ratios in the mature sperm populations. However, among men with immature sperm samples with late spermiogenetic defect and diminished CK-M ratios, there will be two groups: one which completed spermatogenesis prior to spermiogenetic failure (normal LDH-X and diminished CK-M ratios), and another group with defects in both spermatogenesis and spermiogenesis (low LDH-X and diminished CK-M ratios). Because of this heterogeneity, LDH-X ratios will be a poor predictor of sperm maturity. The data support the hypotheses: (1) LDH and CK concentrations were higher in oligospermic vs. normospermic men (P < 0.001). (2) The LDH and CK concentrations were related (r = 0.65, P < 0.001, N = 145), and there were inverse correlations between CK, LDH, LDH-X, or CK-M ratios vs. sperm concentrations (P < 0.001 in all four). (3) The CK-M and LDH-X ratios were different between the oligospermic and normospermic groups (P < 0.001), although the means of the LDH-X ratios were narrower (LDH-X:1:1.3; CK-M:1:1.9). (4) Dividing the 145 samples by the cut-off value of mean minus 1 SD of the CK-M and LDH-X ratios (11% and 32%, respectively) demonstrated that the CK-M ratios discriminated better than LDH-X ratios between the samples with mature and immature sperm. These data on the biochemical markers of early and late spermatogenesis support the studies in which CK better reflected sperm quality than LDH or LDH-X (Orlando et al., 1994: Int J Androl 17:13-18) and the > 10% sperm CK-M ratio predicted with a rate of 30.4% per cycle in the occurrence of pregnancies in a blinded study of 84 IVF couples (Huszar et al., 1992: Fertil Steril 57:882-888).

Structure of myosin heavy chain in avian muscular dystrophy.

We have studied the structure of myosin heavy chain (MHC) in the pectoralis muscle of genetically dystrophic (Connecticut Strain) and White Leghorn chicks. MHC was alkylated with N-ethylmaleimide, purified by Sepharose-4B chromatography, and cleaved with cyanogen bromide. The MHC CNBr peptides were analyzed by one-dimensional and two-dimensional isoelectric focusing/sodium dodecyl sulfate gradient gels and by amino acid sequencing. Specific changes were detected in the gel patterns which could be correlated with the loss of muscle function as measured by the exhaustion score (the ability of chicks to rise from a reclining position) in three experimental groups (exhaustion scores: less than 3, 10-20, greater than 30). We have also examined the amino acid sequence of a 3-methyl-histidine-containing peptide which originates from the 20-kDa fragment of pectoralis muscle MHC in dystrophic chicks: Val-Leu-Asn-Ala-Ser-Ala-Ile-Pro-Glu-Gly-*Gln-Phe-*Ile-Asp-Ser-Lys-Lys- Ala-Ser-Leu-Gln-Lys-Leu-Gly-Ser-Ile-Asp-Val-(Asp, 3-methylhistidine, Gln). Comparison of the homologous MHC sequences shows two positions at which MHC from dystrophic chicks differs from that of the White Leghorn chicks *(Glu----Gln and Met----Ile). Thus, both the peptide map and sequence analyses demonstrate that in avian muscular dystrophy an abnormal pectoralis MHC is synthesized. It is not yet clear whether the "dystrophic" MHC is a variant MHC or if it arises from the abnormal expression of an earlier developmental form (embryonic or neonatal) of pectoralis muscle MHC.

Y-chromosome length in sublines of two mouse strains.

Differences in intermale aggression have been repeatedly reported for DBA/1 and C57BL/10 mice. The results of rreciprocal crosses combined with cross-fostering procedures suggest an involvement of the Y chromosome. In the present study, the length of the Y chromosome relative to that of chromosome 19 was ascertained in five sublines of DBA/1Bg, three sublines of C57BL/10Bg, and C57BL/10.DBA/1-Y congenic stock of mice, which carries the DBA/1Bg Y chromosome. With respect to the length of the Y chromosome relative to that of chromosome of 19, two of the DBA/1 sublines are shorter than the other three DBA/1 sublines, and all DBA/1 sublines are shorter than the three C57BL/10 sublines. This is attributable primarily to the length of the Y chromosome. The C57BL/10 sublines and the BL10.D1-Y congenic stock tested exhibit the same relative lengths of the Y chromosome, suggesting that its length has changed on the C57BL/10 genetic background. There is a parallel dependence on autosomal background of the effect of the Y chromosome on intermale aggression.

FISH assessment of aneuploidy frequencies in mature and immature human spermatozoa classified by the absence or presence of cytoplasmic retention.

Previously, a relationship has been found between diminished cellular maturity of human spermatozoa and low-level expression of the testis-specific chaperone protein, HspA2. Because HspA2 is a component of the synaptonemal complex in rodents, and assuming that this is also the case in men, it was postulated that the frequency of chromosomal aneuploidies would be higher in immature versus mature spermatozoa. This question was examined in spermatozoa from semen and from 80% Percoll pellets (enriched for mature spermatozoa) of the same ejaculate in 10 oligozoospermic men. Immature spermatozoa with retained cytoplasm, which signifies spermiogenetic arrest, were identified by immunocytochemistry. Using fluorescence in-situ hybridization (FISH), approximately 7000 sperm nuclei were evaluated in each of the 20 fractions (142 086 spermatozoa in all) using centromeric probes for the X, Y and 17 chromosomes. The proportions of immature spermatozoa were 45.4 +/- 3.4 versus 26.6 +/- 2.2% in the two semen versus the Percoll groups (medians: 48.2 versus 25%, P < 0.001, n = 300 spermatozoa per fraction, total 6000 spermatozoa). There was also a concomitant decline in total disomy, total diploidy and total aneuploidy frequencies in the 80% Percoll versus semen fractions (0.17 versus 0.54%, 0.14 versus 0.26% and 0.31 versus 0.81% respectively, P < 0.001 in all comparisons). The mean decline of aneuploidies was 2.7-fold. With regard to the hypothesis that aneuploidies are related to sperm immaturity, there was a close correlation between the incidence of immature spermatozoa and disomies (r = 0.7, P < 0.001) but no correlation with diploidies (r = 0.03), indicating that disomies originate primarily in immature spermatozoa. It is suggested that the common factor underlying sperm immaturity and aneuploidies is the diminished expression of HspA2. In addition, the lack of this chaperone may also cause diminished cellular transport of proteins, such as DNA-repair enzymes or of the retention of cytoplasm that is extruded from normally maturing spermatozoa during spermiogenesis.