DECONDENSATION OF HUMAN SPERM NUCLEI AND H P l PROTAMINE DEGRADATION FROM NORMOSPERMIA AND ASTHENOSPERMIA IN XENOPUS EGG EXTRACTS
Syst Biol Reprod Med Downloaded from informahealthcare.com by Queen's University on 12/29/14 For personal use only.
J . F. GRIVEAU, M. CHARBONNEAU, Y. BLANCHARD, D . LESCOAT, and D. LE LANNOU
The process of human sperm decondensation has been studied in vitro in cytoplasmic extracts prepared from unfertilized Xenopus laevis eggs. The chromatin decondensation-recondensation cycle was divided into four stages according to chromatin appearance. Spermatozoa from normospermia and asthenospermia were evaluated according to their capacity to reach these stages, and their DNA integrity was assessed by acridine orange (AO) staining. We observed a significant difference between normospermia and asthenozoospermia in the ability to achieve the cycle of chromatin decondensationrecondensation. These results correlated with A 0 staining. The role of human protamine 1 degradation in the decondensation process was evaluated by immunostaining. It was found not to be a prerequisite for the earlier stage of chromatin decondensation and it was not implied in the latest stages of pronuclear development.
Key Words: Human spermatozoa; Xenopus: Nucleus; Acridine orange.
INTRODUCTION Male fertility is conventionally assessed on the basis of a semen profile reflecting the quality of the ejaculate. However, human spermatozoa, compared to those from other mammalian species, are characterized by a great heterogeneity in their morphology, motility, and nuclear maturity [ 1, 8, 91, making it difficult to predict fertility from semen quality. Some infertile men exhibit a normal semen analysis, so other semen parameters, such as the nuclear decondensation-recondensation process during fertilization, may be involved in male fertility. The transformation of the nucleus of the spermatozoa into a pronucleus is a complex process during which the sperm nucleus, activated by the egg cytoplasm, undergoes a series of changes restoring its functions, which are nuclear envelope breakdown, chromatin decon-
From the CECOS de I’ouest, Hotel-Dieu (J.F.G., Y.B., D.L.L.); Laboratoire de biologie et gCnetique du diveloppement, Universite de Rennes I (M.C., D.L.L.); and Unite de biologie de la reproduction, laboratoire d’histologie et d’embryologie, Rennes, France (D.L., D.L.L.). Address correspondence to J . E Griveau, CECOS de l’ouest, Hotel-Dieu, 1 bis rue de la cocharditre, 35000, Rennes, France. ARCHIVES OF ANDROLOGY 29: 127-136 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
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Syst Biol Reprod Med Downloaded from informahealthcare.com by Queen's University on 12/29/14 For personal use only.
J. F. Griveau et al. densation, replacement of nucleoprotamines by histones, and DNA synthesis [ 131. Analysis of these processes is difficult in humans because of an extremely limited amount of biological material available for biochemical research. So in the present study, we have used the egg from the amphibian Xenopus laevis, a heterospecific system that can mimic i n vitro some of the early events of fertilization [ 111. To evaluate the possible relation between the semen characteristics and the process of pronuclear formation, we have conducted an investigation of the main structural changes during the decondensation of human sperm nuclei, and we have evaluated their D N A integrity and the timing of HP1 protamine degradation. The study was performed on ejaculated spermatozoa obtained from normospermia and asthenozoospermia.
MATERIALS AND METHODS Egg Extract Preparation. Egg cytoplasmic extracts were prepared according to Lohka and Maller [12]. Adult female Xenopus luevis were induced to ovulate following injection with 900 IU of human chorionic gonadotrophin. Mature oviposited eggs, arrested at the second metaphase of meiosis, were immersed in F1 solution (31.2 mM NaCl, 1.8 mM KCI, 1.0 mM CaCI,, 1.9 mM NaOH, 10.0 mM Hepes, 2.0 mM NaHCO,) containing 2 % cysteine, pH 7.8, and gently swirled for about 10 min to remove the jelly layers. After rinsing three times in tap water supplemented with 110 mM NaCl, dejellied eggs were transferred to 75 mL extraction buffer containing 100 mM KCI, 5 mM MgCl,, 20 mM Hepes, pH 7.5, and supplemented before use with 0.3 mM phenylmethylsulfonyl fluoride and 3 pg/mL leupeptin. Excess buffer was carefully removed with a Pasteur pipette and the eggs were lysed by centrifugation (lO,OOOg, 10 min) at 4°C. The supernatant, between lipid and yolk layers was collected, supplemented with cytochalasin B at a final concentration of 50 pg/mL, and then recentrifuged (lO,OOOg, 10 min, 4°C). The resulting supernatant was collected, transferred into Eppendorf tubes, and supplemented with aprotinin (0.8 IU/mL) and leupeptin (6 pg/mL). All supernatants were pooled and stored at - 196°C. Sperm Preparation. Human semen was obtained by masturbation after a 3-day period of sexual abstinence and allowed to liquefy at 37 "C for 30 min. Semen parameters, including volume, number, motility, and morphology, were measured and two groups were defined: Group 1, normospermic samples from candidates for vasectomy with >20 X lo6 spermatozoa/mL, >50% motility; and group 2, asthenozoospermic samples from men consulting for sterility with > 20 x lo6 spermatozoa/ mL,
Syst Biol Reprod Med Downloaded from informahealthcare.com by Queen's University on 12/29/14 For personal use only.
J . F. GRIVEAU, M. CHARBONNEAU, Y. BLANCHARD, D . LESCOAT, and D. LE LANNOU
The process of human sperm decondensation has been studied in vitro in cytoplasmic extracts prepared from unfertilized Xenopus laevis eggs. The chromatin decondensation-recondensation cycle was divided into four stages according to chromatin appearance. Spermatozoa from normospermia and asthenospermia were evaluated according to their capacity to reach these stages, and their DNA integrity was assessed by acridine orange (AO) staining. We observed a significant difference between normospermia and asthenozoospermia in the ability to achieve the cycle of chromatin decondensationrecondensation. These results correlated with A 0 staining. The role of human protamine 1 degradation in the decondensation process was evaluated by immunostaining. It was found not to be a prerequisite for the earlier stage of chromatin decondensation and it was not implied in the latest stages of pronuclear development.
Key Words: Human spermatozoa; Xenopus: Nucleus; Acridine orange.
INTRODUCTION Male fertility is conventionally assessed on the basis of a semen profile reflecting the quality of the ejaculate. However, human spermatozoa, compared to those from other mammalian species, are characterized by a great heterogeneity in their morphology, motility, and nuclear maturity [ 1, 8, 91, making it difficult to predict fertility from semen quality. Some infertile men exhibit a normal semen analysis, so other semen parameters, such as the nuclear decondensation-recondensation process during fertilization, may be involved in male fertility. The transformation of the nucleus of the spermatozoa into a pronucleus is a complex process during which the sperm nucleus, activated by the egg cytoplasm, undergoes a series of changes restoring its functions, which are nuclear envelope breakdown, chromatin decon-
From the CECOS de I’ouest, Hotel-Dieu (J.F.G., Y.B., D.L.L.); Laboratoire de biologie et gCnetique du diveloppement, Universite de Rennes I (M.C., D.L.L.); and Unite de biologie de la reproduction, laboratoire d’histologie et d’embryologie, Rennes, France (D.L., D.L.L.). Address correspondence to J . E Griveau, CECOS de l’ouest, Hotel-Dieu, 1 bis rue de la cocharditre, 35000, Rennes, France. ARCHIVES OF ANDROLOGY 29: 127-136 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation
127
Syst Biol Reprod Med Downloaded from informahealthcare.com by Queen's University on 12/29/14 For personal use only.
J. F. Griveau et al. densation, replacement of nucleoprotamines by histones, and DNA synthesis [ 131. Analysis of these processes is difficult in humans because of an extremely limited amount of biological material available for biochemical research. So in the present study, we have used the egg from the amphibian Xenopus laevis, a heterospecific system that can mimic i n vitro some of the early events of fertilization [ 111. To evaluate the possible relation between the semen characteristics and the process of pronuclear formation, we have conducted an investigation of the main structural changes during the decondensation of human sperm nuclei, and we have evaluated their D N A integrity and the timing of HP1 protamine degradation. The study was performed on ejaculated spermatozoa obtained from normospermia and asthenozoospermia.
MATERIALS AND METHODS Egg Extract Preparation. Egg cytoplasmic extracts were prepared according to Lohka and Maller [12]. Adult female Xenopus luevis were induced to ovulate following injection with 900 IU of human chorionic gonadotrophin. Mature oviposited eggs, arrested at the second metaphase of meiosis, were immersed in F1 solution (31.2 mM NaCl, 1.8 mM KCI, 1.0 mM CaCI,, 1.9 mM NaOH, 10.0 mM Hepes, 2.0 mM NaHCO,) containing 2 % cysteine, pH 7.8, and gently swirled for about 10 min to remove the jelly layers. After rinsing three times in tap water supplemented with 110 mM NaCl, dejellied eggs were transferred to 75 mL extraction buffer containing 100 mM KCI, 5 mM MgCl,, 20 mM Hepes, pH 7.5, and supplemented before use with 0.3 mM phenylmethylsulfonyl fluoride and 3 pg/mL leupeptin. Excess buffer was carefully removed with a Pasteur pipette and the eggs were lysed by centrifugation (lO,OOOg, 10 min) at 4°C. The supernatant, between lipid and yolk layers was collected, supplemented with cytochalasin B at a final concentration of 50 pg/mL, and then recentrifuged (lO,OOOg, 10 min, 4°C). The resulting supernatant was collected, transferred into Eppendorf tubes, and supplemented with aprotinin (0.8 IU/mL) and leupeptin (6 pg/mL). All supernatants were pooled and stored at - 196°C. Sperm Preparation. Human semen was obtained by masturbation after a 3-day period of sexual abstinence and allowed to liquefy at 37 "C for 30 min. Semen parameters, including volume, number, motility, and morphology, were measured and two groups were defined: Group 1, normospermic samples from candidates for vasectomy with >20 X lo6 spermatozoa/mL, >50% motility; and group 2, asthenozoospermic samples from men consulting for sterility with > 20 x lo6 spermatozoa/ mL,
