A new method of cell fusion is proposed utilizing treatment with 15% solution of DMSO in serum be... more A new method of cell fusion is proposed utilizing treatment with 15% solution of DMSO in serum before and after PEG treatment. With such treatments in SPEV cell culture a higher rate of cell fusion was obtained than that with other known methods of cell fusion. In the first wave of mitoses (0.5-4 h) mainly asynchronous division of nuclei, premature chromosome condensation and formation of telophase-like nuclei were observed in polykaryons. In the period of the second wave (14-20 h), mitoses were mainly synchronous and completed with cytokinesis. Micronuclei were formed frequently as a result of such mitoses. After the first wave of mitoses the number of polykaryons with pycnotic chromosomes sharply increased, and after the second wave of mitoses the number of polykaryons with pycnotic nuclei increased. The results obtained allow to conclude that heterophasic condition of the fused cells is one of the causes of pathological mitosis of polykaryons and of their death.
Publisher Summary Spermiogenesis is the terminal differentiation of postmeiotic germ cells into s... more Publisher Summary Spermiogenesis is the terminal differentiation of postmeiotic germ cells into spermatozoa. The cellular and molecular pathways of centrosome reduction during mouse, rhesus, and human spermiogenesis are described. This chapter discusses the fundamental events and stages of centrosome reduction during mammalian spermiogenesis. The developmental stages of isolated mouse spermatids show remarkable events of centrosomal changes: round spermatid stage, elongating spermatid stage, and late elongating stage. The process of centrosome reduction extends throughout the entire testicular and epididymal stages of spermiogenesis and comprises four stages: (a) loss of microtubule nucleating function, (b) formation of transitional microtubule organizing centers (MTOCs), (c) loss of centrosomal proteins, and (d) centriole degeneration. The observation of a systematic degeneration of distal centriole and γ-tubulin in nonrodent mammalian sperm implies that centrosome reduction could be a ubiquitous phenomenon taking place in all mammalian sperm to varying degrees, the mouse sperm representing the highest state and exhibiting complete loss of both centrioles and centrosomal proteins. The complete absence of centrosomes in mouse sperm consolidates the hypothesis that murine oocytes can compensate for the lack of paternal centrosomal contribution. The lack of a standard distal centriole and γ-tubulin in rhesus and human sperm does not interfere with the formation of the zygotic centrosome during fertilization and bipolar spindles during cleavages.
The protein gene product 9.5 (PGP9.5) belongs to a family of ubiquitin C-terminal hydrolases (UCH... more The protein gene product 9.5 (PGP9.5) belongs to a family of ubiquitin C-terminal hydrolases (UCHs), which regenerate monoubiquitin from ubiquitin–protein complexes or polyubiquitin chains by cleaving the amide linkage next to the C-terminal glycine of ubiquitin. Identified in the acrosome of boar spermatozoa, we hypothesized that PGP9.5 might regulate sperm–zona pellucida interactions during porcine IVF. The cumulus–oocyte complexes isolated from slaughterhouse ovaries were cultured in TCM-199 media for 44 h at 38.5�C, 5% CO2 in air. After completion of in vitro maturation (IVM), cumulus cells were removed by 0.1% hyaluronidase, and metaphase II (MII) oocytes were used for IVF. In Experiment 1, oocytes were co-incubated with different sperm concentrations (1 � 106, 5 � 105, and 1 � 105 sperm mL-1) in TBM medium with or without anti-PGP9.5 antibody (1 : 50 dilution). In Experiment 2, oocytes were inseminated with 1 � 106 sperm mL-1 in TBM medium containing different concentrations of extracted oviductal fluids (0, 0.1, 0.5, 1, 2, and 3 �g mL-1) for 6 h. After IVF, oocytes were transferred into NCSU23 medium containing 0.4% BSA for further culture. The fertilization rates were evaluated by DAPI staining at 13 to 19 h. Data were analyzed by ANOVA and Duncan's multiple range test using the SAS program. Polyspermy was increased by the addition of anti-PGP9.5 antibody to the IVF medium (56.5–60.2% at polyspermy). This PGP9.5-antibody-induced polyspermy increase was sustained even with decreasing sperm concentrations. The polyspermy rates were reduced by the addition of isolated porcine oviductal fluid to IVF medium (50.4, 44.8, 28.0, 31.1, 1.6, and 0.0% at oviductal fluid concentrations of 0, 0.1, 0.5, 1, 2, and 3 µg mL−1, respectively). Biochemical analysis by Western blotting detected the appropriate 24-kDa PGP9.5 band in porcine oviductal fluid used for these experiments. Enzymatic UCH activity comparable to activity of recombinant UCH-L3 was detected in sperm extract, whole spermatozoa, and isolated oviductal fluid by fluorometric assay using fluorogenic UCH-substrate ubiquitin-AMC. This UCH activity was not reduced by the general protease inhibitor phenyl methyl sylfonyl fluoride, but it was reduced in a statistically significant manner (P < 0.05) by the specific UCH-inhibitor ubiquitin aldehyde. In conclusion, the polyspermy increased with different concentrations of sperm in the anti-PGP9.5 antibody, and PGP9.5 was detected in oviductal fluids, suggesting that PGP9.5 is involved in the sperm–zona pellucida interaction during porcine fertilization. This work was supported by the US Department of Agriculture (USDA-NRI grant #2002-35203-12237 to P.S.), the F21C Program of The University of Missouri–Columbia (P.S.), and the ERC Program of the Korea Science and Engineering Foundation (KOSEF grant no. R11-2002-100-00000-0 to Y.J. Yi and C.S. Park).
Molecular Reproduction and Development, Aug 1, 2000
Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-cen... more Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-centrin antibodies and electron microscopy in nonhuman primates. Rhesus spermatids possess apparently normal centrosomes comprising a pair of centrioles associated with gamma-tubulin and centrin. However, they do not nucleate detectable microtubules. The spermatids discard gamma-tubulin in the residual bodies during the spermiation stage. Mature sperm do not have any detectable gamma-tubulin. About half of the centrin associated with the distal centriole degenerates during spermiogenesis and the remainder is intimately bound to the centriolar microtubules. The mature sperm possess highly degenerated distal centrioles. The centriolar microtubules degenerate in the rostral region and the ventral side of the sperm. The study indicates that the centrosome is reduced during rhesus spermiogenesis, but not completely as in mice.
Mammals have two protamine genes that encode for protamine 1 and protamine 2. Due to the presence... more Mammals have two protamine genes that encode for protamine 1 and protamine 2. Due to the presence of extensive, stable cross-linking by SS bonds, the sperm chromatin is protected from damage by the external physical or chemical disruptive agents. However, ...
What is said above argues that the male contribution at fertilization is restricted to one half o... more What is said above argues that the male contribution at fertilization is restricted to one half of future embryonic chromosomes. Although inspired by general perception and often perpetuated by popular science, this assumption is incorrect. Besides being a launching ...
Mammalian spermatozoa complete their morphogenesis and acquire their fertilizing potential in the... more Mammalian spermatozoa complete their morphogenesis and acquire their fertilizing potential in the epididymis. Prominent among the hallmarks of epididymal sperm maturation is the proximal-distal migration of the cytoplasmic droplet (CD), the last remnant of the spermatogenic cell cytoplasm, down the sperm flagellum. Failure to shed the CD has been associated with male infertility. Because of the presence of the organelle degradation enzyme 15-lipoxygenase (15LOX) in sperm CD, we hypothesize that subfertile male Alox15 mice lacking the 15Lox gene display sperm CD anomalies. Caput and cauda epididymal sperm samples from seven adult Alox15 and seven wild-type (wt) males of equal age were examined by differential interference contrast microscopy (DIC) and transmission electron microscopy (TEM). Compared with wt males, Alox15 males had significantly more spermatozoa with a retained CD in both caput (P = 0.004) and cauda (P = 0.005) epididymidis. TEM and DIC analyses revealed intact mitochondria present in the CDs of epididymal Alox15 spermatozoa. The CDs of wt spermatozoa, however, had a smooth appearance and contained only hollow membrane vesicles, with no intact mitochondria embedded in their CD matrix. Epithelial lesions, phagocytosis-like figures, and missing or aberrant apical blebs were observed in the caput epididymidis of Alox15 males. Thus, the process of epididymal sperm maturation and CD migration is altered in Alox15 males. Aberrant sperm maturation might contribute to the reduced fertility and smaller litter size of Alox15 mice, a rare example of subfertile mutants displaying normal spermatogenesis but altered epididymal sperm maturation.
The purpose of this study was to investigate the relationship between fertility and quantitative ... more The purpose of this study was to investigate the relationship between fertility and quantitative measures of boar semen quality, including various patterns of sperm cytoplasmic droplet (CD) retention, as determined by high power differential interference contrast (DIC) microscopy. A total of 116 ejaculates were collected from a nucleus herd of 18 Large White boars over an eight month period. Semen quality parameters were analyzed for each ejaculate by calculating the percentage of normal spermatozoa, spermatozoa possessing a CD in the proximal, distal, or distal midpiece reflex position, total spermatozoa with an attached cytoplasmic droplet, spermatozoa with non-CD related aberrations and total spermatozoa with abnormalities. Of the 116 ejaculates received, 71 ejaculates from 13 boars had corresponding fertility data from single-sire inseminations of multiparous sows. The fertility data included farrowing rate (FR) and total number born (TNB). The monthly FR encompassed one month before and one month after the date of semen collection. Detection of differences for fertility and semen quality parameters was performed by separating the boars into either an above-average or below-average group based on the mean FR (74.01 +/- 1.43%) or TNB (12.34 +/- 0.17) for the study. For FR, the boars in the below-average group had a significantly lower percentage of normal spermatozoa and significantly higher percentage of spermatozoa possessing distal CDs, total attached CDs and total abnormalities compared to the boars in the above-average group. Conversely, for TNB there were no significant differences between the above- and below-average groups for the semen quality parameters. These data suggest that the attached CD may negatively affect FR, but not TNB. The detection of relationships between the boar fertility parameters and the retention of the sperm CD after ejaculation, document the advantage of high power DIC microscopy in conventional semen evaluation.
Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-cen... more Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-centrin antibodies and electron microscopy in nonhuman primates. Rhesus spermatids possess apparently normal centrosomes comprising a pair of centrioles associated with gamma-tubulin and centrin. However, they do not nucleate detectable microtubules. The spermatids discard gamma-tubulin in the residual bodies during the spermiation stage. Mature sperm do not have any detectable gamma-tubulin. About half of the centrin associated with the distal centriole degenerates during spermiogenesis and the remainder is intimately bound to the centriolar microtubules. The mature sperm possess highly degenerated distal centrioles. The centriolar microtubules degenerate in the rostral region and the ventral side of the sperm. The study indicates that the centrosome is reduced during rhesus spermiogenesis, but not completely as in mice.
A new method of cell fusion is proposed utilizing treatment with 15% solution of DMSO in serum be... more A new method of cell fusion is proposed utilizing treatment with 15% solution of DMSO in serum before and after PEG treatment. With such treatments in SPEV cell culture a higher rate of cell fusion was obtained than that with other known methods of cell fusion. In the first wave of mitoses (0.5-4 h) mainly asynchronous division of nuclei, premature chromosome condensation and formation of telophase-like nuclei were observed in polykaryons. In the period of the second wave (14-20 h), mitoses were mainly synchronous and completed with cytokinesis. Micronuclei were formed frequently as a result of such mitoses. After the first wave of mitoses the number of polykaryons with pycnotic chromosomes sharply increased, and after the second wave of mitoses the number of polykaryons with pycnotic nuclei increased. The results obtained allow to conclude that heterophasic condition of the fused cells is one of the causes of pathological mitosis of polykaryons and of their death.
Publisher Summary Spermiogenesis is the terminal differentiation of postmeiotic germ cells into s... more Publisher Summary Spermiogenesis is the terminal differentiation of postmeiotic germ cells into spermatozoa. The cellular and molecular pathways of centrosome reduction during mouse, rhesus, and human spermiogenesis are described. This chapter discusses the fundamental events and stages of centrosome reduction during mammalian spermiogenesis. The developmental stages of isolated mouse spermatids show remarkable events of centrosomal changes: round spermatid stage, elongating spermatid stage, and late elongating stage. The process of centrosome reduction extends throughout the entire testicular and epididymal stages of spermiogenesis and comprises four stages: (a) loss of microtubule nucleating function, (b) formation of transitional microtubule organizing centers (MTOCs), (c) loss of centrosomal proteins, and (d) centriole degeneration. The observation of a systematic degeneration of distal centriole and γ-tubulin in nonrodent mammalian sperm implies that centrosome reduction could be a ubiquitous phenomenon taking place in all mammalian sperm to varying degrees, the mouse sperm representing the highest state and exhibiting complete loss of both centrioles and centrosomal proteins. The complete absence of centrosomes in mouse sperm consolidates the hypothesis that murine oocytes can compensate for the lack of paternal centrosomal contribution. The lack of a standard distal centriole and γ-tubulin in rhesus and human sperm does not interfere with the formation of the zygotic centrosome during fertilization and bipolar spindles during cleavages.
The protein gene product 9.5 (PGP9.5) belongs to a family of ubiquitin C-terminal hydrolases (UCH... more The protein gene product 9.5 (PGP9.5) belongs to a family of ubiquitin C-terminal hydrolases (UCHs), which regenerate monoubiquitin from ubiquitin–protein complexes or polyubiquitin chains by cleaving the amide linkage next to the C-terminal glycine of ubiquitin. Identified in the acrosome of boar spermatozoa, we hypothesized that PGP9.5 might regulate sperm–zona pellucida interactions during porcine IVF. The cumulus–oocyte complexes isolated from slaughterhouse ovaries were cultured in TCM-199 media for 44 h at 38.5�C, 5% CO2 in air. After completion of in vitro maturation (IVM), cumulus cells were removed by 0.1% hyaluronidase, and metaphase II (MII) oocytes were used for IVF. In Experiment 1, oocytes were co-incubated with different sperm concentrations (1 � 106, 5 � 105, and 1 � 105 sperm mL-1) in TBM medium with or without anti-PGP9.5 antibody (1 : 50 dilution). In Experiment 2, oocytes were inseminated with 1 � 106 sperm mL-1 in TBM medium containing different concentrations of extracted oviductal fluids (0, 0.1, 0.5, 1, 2, and 3 �g mL-1) for 6 h. After IVF, oocytes were transferred into NCSU23 medium containing 0.4% BSA for further culture. The fertilization rates were evaluated by DAPI staining at 13 to 19 h. Data were analyzed by ANOVA and Duncan's multiple range test using the SAS program. Polyspermy was increased by the addition of anti-PGP9.5 antibody to the IVF medium (56.5–60.2% at polyspermy). This PGP9.5-antibody-induced polyspermy increase was sustained even with decreasing sperm concentrations. The polyspermy rates were reduced by the addition of isolated porcine oviductal fluid to IVF medium (50.4, 44.8, 28.0, 31.1, 1.6, and 0.0% at oviductal fluid concentrations of 0, 0.1, 0.5, 1, 2, and 3 µg mL−1, respectively). Biochemical analysis by Western blotting detected the appropriate 24-kDa PGP9.5 band in porcine oviductal fluid used for these experiments. Enzymatic UCH activity comparable to activity of recombinant UCH-L3 was detected in sperm extract, whole spermatozoa, and isolated oviductal fluid by fluorometric assay using fluorogenic UCH-substrate ubiquitin-AMC. This UCH activity was not reduced by the general protease inhibitor phenyl methyl sylfonyl fluoride, but it was reduced in a statistically significant manner (P < 0.05) by the specific UCH-inhibitor ubiquitin aldehyde. In conclusion, the polyspermy increased with different concentrations of sperm in the anti-PGP9.5 antibody, and PGP9.5 was detected in oviductal fluids, suggesting that PGP9.5 is involved in the sperm–zona pellucida interaction during porcine fertilization. This work was supported by the US Department of Agriculture (USDA-NRI grant #2002-35203-12237 to P.S.), the F21C Program of The University of Missouri–Columbia (P.S.), and the ERC Program of the Korea Science and Engineering Foundation (KOSEF grant no. R11-2002-100-00000-0 to Y.J. Yi and C.S. Park).
Molecular Reproduction and Development, Aug 1, 2000
Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-cen... more Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-centrin antibodies and electron microscopy in nonhuman primates. Rhesus spermatids possess apparently normal centrosomes comprising a pair of centrioles associated with gamma-tubulin and centrin. However, they do not nucleate detectable microtubules. The spermatids discard gamma-tubulin in the residual bodies during the spermiation stage. Mature sperm do not have any detectable gamma-tubulin. About half of the centrin associated with the distal centriole degenerates during spermiogenesis and the remainder is intimately bound to the centriolar microtubules. The mature sperm possess highly degenerated distal centrioles. The centriolar microtubules degenerate in the rostral region and the ventral side of the sperm. The study indicates that the centrosome is reduced during rhesus spermiogenesis, but not completely as in mice.
Mammals have two protamine genes that encode for protamine 1 and protamine 2. Due to the presence... more Mammals have two protamine genes that encode for protamine 1 and protamine 2. Due to the presence of extensive, stable cross-linking by SS bonds, the sperm chromatin is protected from damage by the external physical or chemical disruptive agents. However, ...
What is said above argues that the male contribution at fertilization is restricted to one half o... more What is said above argues that the male contribution at fertilization is restricted to one half of future embryonic chromosomes. Although inspired by general perception and often perpetuated by popular science, this assumption is incorrect. Besides being a launching ...
Mammalian spermatozoa complete their morphogenesis and acquire their fertilizing potential in the... more Mammalian spermatozoa complete their morphogenesis and acquire their fertilizing potential in the epididymis. Prominent among the hallmarks of epididymal sperm maturation is the proximal-distal migration of the cytoplasmic droplet (CD), the last remnant of the spermatogenic cell cytoplasm, down the sperm flagellum. Failure to shed the CD has been associated with male infertility. Because of the presence of the organelle degradation enzyme 15-lipoxygenase (15LOX) in sperm CD, we hypothesize that subfertile male Alox15 mice lacking the 15Lox gene display sperm CD anomalies. Caput and cauda epididymal sperm samples from seven adult Alox15 and seven wild-type (wt) males of equal age were examined by differential interference contrast microscopy (DIC) and transmission electron microscopy (TEM). Compared with wt males, Alox15 males had significantly more spermatozoa with a retained CD in both caput (P = 0.004) and cauda (P = 0.005) epididymidis. TEM and DIC analyses revealed intact mitochondria present in the CDs of epididymal Alox15 spermatozoa. The CDs of wt spermatozoa, however, had a smooth appearance and contained only hollow membrane vesicles, with no intact mitochondria embedded in their CD matrix. Epithelial lesions, phagocytosis-like figures, and missing or aberrant apical blebs were observed in the caput epididymidis of Alox15 males. Thus, the process of epididymal sperm maturation and CD migration is altered in Alox15 males. Aberrant sperm maturation might contribute to the reduced fertility and smaller litter size of Alox15 mice, a rare example of subfertile mutants displaying normal spermatogenesis but altered epididymal sperm maturation.
The purpose of this study was to investigate the relationship between fertility and quantitative ... more The purpose of this study was to investigate the relationship between fertility and quantitative measures of boar semen quality, including various patterns of sperm cytoplasmic droplet (CD) retention, as determined by high power differential interference contrast (DIC) microscopy. A total of 116 ejaculates were collected from a nucleus herd of 18 Large White boars over an eight month period. Semen quality parameters were analyzed for each ejaculate by calculating the percentage of normal spermatozoa, spermatozoa possessing a CD in the proximal, distal, or distal midpiece reflex position, total spermatozoa with an attached cytoplasmic droplet, spermatozoa with non-CD related aberrations and total spermatozoa with abnormalities. Of the 116 ejaculates received, 71 ejaculates from 13 boars had corresponding fertility data from single-sire inseminations of multiparous sows. The fertility data included farrowing rate (FR) and total number born (TNB). The monthly FR encompassed one month before and one month after the date of semen collection. Detection of differences for fertility and semen quality parameters was performed by separating the boars into either an above-average or below-average group based on the mean FR (74.01 +/- 1.43%) or TNB (12.34 +/- 0.17) for the study. For FR, the boars in the below-average group had a significantly lower percentage of normal spermatozoa and significantly higher percentage of spermatozoa possessing distal CDs, total attached CDs and total abnormalities compared to the boars in the above-average group. Conversely, for TNB there were no significant differences between the above- and below-average groups for the semen quality parameters. These data suggest that the attached CD may negatively affect FR, but not TNB. The detection of relationships between the boar fertility parameters and the retention of the sperm CD after ejaculation, document the advantage of high power DIC microscopy in conventional semen evaluation.
Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-cen... more Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-centrin antibodies and electron microscopy in nonhuman primates. Rhesus spermatids possess apparently normal centrosomes comprising a pair of centrioles associated with gamma-tubulin and centrin. However, they do not nucleate detectable microtubules. The spermatids discard gamma-tubulin in the residual bodies during the spermiation stage. Mature sperm do not have any detectable gamma-tubulin. About half of the centrin associated with the distal centriole degenerates during spermiogenesis and the remainder is intimately bound to the centriolar microtubules. The mature sperm possess highly degenerated distal centrioles. The centriolar microtubules degenerate in the rostral region and the ventral side of the sperm. The study indicates that the centrosome is reduced during rhesus spermiogenesis, but not completely as in mice.
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