D e p a r t m a t s of Obstetrics cirid Gyneroloqy rind A n u t o m y , Cornell Lrn~vehzty Medzcal College, Nezu York, h e w York 1002a n d 2 Internnt7onulI n s f z t r i t P oi H u m a n Rpproductzon, C o ABSTRACTThe extraordinary resistance to disruption shown by the eutherian sperm nucleus is associated with a significant complement of -S-S-crosslinks within the chromatin. The present light and electron microscopic study, in which the spermatozoa of many vertebrates and some invertebrates were exposed to SDS writh or without dithiothreitol, demonstrates that the extreme degree of disulfide bonding in the sperm chromatin of eutherian mammals is highly unusual in a comparative sense. In no other species did the sperm nuclcus display a resistance to disruption comparable to that in eutherians; on the contrary, in most groups including prototherian and metatherian mammals, such crosslinks are absent or relatively sparse, the greatest degree of crosslinking outside the eutheria being seen in octopus and skate.The suggestion is advanced that the unusually thiol-rich protamines have evolved as a structural adaptation which, through crosslinking. endows the sperm head with a rigidity of possible importance in the penetration phase of fertilization. This seems likely because (1) any degree of -S-S-determined stability present or inducible in sperm chromatin often appears more intense in or is confined to the anterior region of the nucleus (e.g., newt, snail, turtle and immature eutherian spermatozoa); (2) the zona pellucida, which the fertilizing sperm must penetrate, is an unusually thick, compact and resilient coat in the eutherian ovum in particular; and (3) the occasional observation of a sperm nucleus buckled within the pathway of its penetration into the rabbit zona pellucida suggcsts that in eutheria this tough vestment presents a relatively rigid barrier to the sperm head.Its apical location suggests a similar structural role for the -S-S-stabiliscd perinuclear projection, or "perforatorium" around the leading bordcr of the sperm nucleus in eutherians as well as in some non-eutherian species, such as octopus, snake and turtle.It has been known sincc the investigations of Miescher that the dense nucleus of the eutherian spermatozoon is highly resistant to disruption, and its intractable quality is now believed to depend on a significant complement of' disulfide crosslinks within the chromatin '63; Hendricks and Mayer, '65). These bonds occur between half-cysteine residues whose concentration in eutherian protamines rests between 8 and 16% according to the species (Coelingh, Rozijn and Monfoort, '69; Coelingh et '73; Calvin, '74). This is comparable to the cysteine content of keratins (Mercer, '61).After penetration of the sperm into the eutherian egg, formation of the male pronucleus initially involves immediate decondensation of the chromatin into strands of nucleoprotein (Barros and Franklin, '68; Yanagimachi and Noda, '70b; Bedford, '70, '72). Little appears to be known of the mechanism of sperm nu...
Glutathione (GSH), the major low‐molecular‐weight thiol in mammalian cells, is believed to be a necessary factor for the transformation of the disulfide‐stabilized sperm nucleus into the male pronucleus after fertilization. Its concentration in mouse ova, isolated from the ampulla of the oviduct after hormone‐induced superovulation of 3–4‐week‐old mice, has been determined by an enzymic cycling microassay. The level found was 1.80 pmol per ovum. Mean ovum diameter was estimated as 71–72 μm, indicating a GSH concentration of 9–10 mM in the mouse egg. Administration of L‐buthionine S, R‐sulfoximine, an inhibitor of GSH biosynthesis, during the 2 days preceding ovulation, reduced ovum GSH content below 0.20 pmol (<1.0 mM). The mean GSH concentration of the hormone‐stimulated ovaries was reduced from 3.2 mM to 0.2 mM under these conditions. It has also been demonstrated that measurement and manipulation of ovum and ovarian levels of GSH can aid in studying its function in ovaries, ova, and early embryos. Hormone‐induced superovulation was achieved in BSO‐treated prepuberal C57B1/6 X SJL mice whose ovaries contained less than 10% of control levels of GSH. Over 50% of the isolated ova were fertilized in vitro. However, abnormal one‐cell embryos resulted in which the maternally derived pronucleus coexisted with an unchanged sperm nucleus, thus confirming that adequate levels of GSH are necessary for initiating transformation of the fertilizing sperm nucleus.
Treatment of immature and mature epididymal spermatoz,oa with SDS and DTT reveals differences in the structural character of their respective tail organelles as a function of their maturity, which are riot apparent in untreated spermatozoa. The response of mature sperm tails of rat, rabbit and other mammals to these reagents indicates that the segmented connecting pieces. the coarse outer fibers, the outer membrane of the mitochondrion and the fibrous sheath of the principal piece, as well as the relationships between these organelles, are stabilized highly by intermolecular -SScrosslinks. The disruption of these structures by SDS alone in caput epididymal spermatozoa, however, implies their stabilization by disulfide bonds during sperm passage through the epididymis. Hence the pattern of flagellar beat seen typically i n mature epididy-ma1 or ejaculated spermatozoa is probably determined by post-spermiation changes in the structural as well as in the metabolic properties of the tail organellcs.In similar studies of a wide variety of sub-mammalian species, the sperm tails fell into two broad classes. The "simple" tail, a plasma membrane enclosing only mitochondria and microtubule doublets (teleosts, frog, chicken and pigeon), disappeared immediately in SDS, though a partial -%Sdependent stability was inducible in the microtubules of telcost sperm tails. In the second class, the coarse fibers, arrayed as a nine-membered ring (octopus, snake, lizard, turtle and sparrow) or otherwise (skate, newt), and the fibrous sheath (snake, lizard) also are stabilized by 4 -S -bonds, as is the undulating membrane of the urodele sperm tail.Since the sperm mitochondria in all sub-mammalian species were totally disrupted hy SDS alone, an extreme -S-% dependent stability of the outer mitochondrial membrane may well be a feature unique to mammalian spermatozoa.
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