James K. Koehler

Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm.

OBJECTIVE To examine the outcome of intracytoplasmic sperm injection (ICSI) with round-headed sperm (globozoospermia). DESIGN Retrospective analysis. SETTING In vitro fertilization laboratory with extensive ICSI experience. PATIENT(S) A patient couple with infertility because of globozoospermia seeking ICSI treatment. MAIN OUTCOME MEASURE(S) Fertilization, cleavage, and pregnancy rates. INTERVENTION(S) Intracytoplasmic sperm injection and calcium ionophore. RESULT(S) This couple experienced only 7% fertilization after ICSI in their first cycle. Treatment of the unfertilized oocytes with calcium ionophore 20 hours after ICSI-induced fertilization and cleavage of 70% of the oocytes. Embryo quality was fair to good. On the second cycle, 8 of the injected oocytes were treated with ionophore immediately after ICSI and the remaining 20 oocytes were untreated. Normal fertilization was achieved in 75% of the treated and 10% of the untreated oocytes. Treatment of these unfertilized oocytes with ionophore 20 hours after ICSI resulted in fertilization in 73%. Pregnancy was not achieved after either ICSI cycle. Ultrastructural analysis indicated multiple structural abnormalities in the sperm. CONCLUSION(S) These results indicate that the round-headed sperm from this patient were incapable of oocyte activation after ICSI. This may be the reason for the frequent ICSI fertilization failure seen with this condition. Current ICSI procedures may not always overcome the infertility associated with globozoospermia, and further study of the etiology of this condition is needed.

A freeze-etching study of rabbit spermatozoa with particular reference to head structures.

The fine structure of rabbit spermatozoa has been studied using the freeze-etching technique. The postnuclear sheath is revealed as a complex structure consisting of a matrix material ornamented by irregular depressions, basally located cords of oriented particles, and a terminal belt-like structure. The apical border of the postnuclear sheath is delineated by highly regular serrations. Many of these components can also be detected in thin sections of appropriately oriented spermatozoa. The nuclear region is composed of stacked lamellae lying in the plane of the flattened head. These lamellae can also be visualized in chemically fixed thin sections and may be related to the birefringence displayed by these cells under polarized light.

The Mammalian Sperm Surface: Studies with Specific Labeling Techniques

Publisher Summary This chapter focuses on electron microscope studies of spermatozoa, which include specific information concerning distributions of chemical entities on the sperm or acrosomal surface. Of particular interest are studies relating to some of the dynamic transformations, such ascapacitation, the acrosome reaction, and membrane fusion). Although much attention has been directed to some of these aspects in invertebrate systems; this chapter discusses the findings made on mammalian spermatozoa. Information will continue to accumulate with respect to the constellation of specific chemical sites and receptors on the sperm surface over the next few years. Hopefully, patterns will emerge that will be useful in analyzing the biological activities of spermatozoa throughout their life cycle and that will have some general validity beyond particular species characteristics. Of special interest here are capacitation, the acrosome reaction, and sperm-egg interactions. To pursue these ends and have confidence in our observations, one must utilize more effective procedures for maximizing the particular cellular stages for analysis with fine-structural techniques. Some methods for synchronizing the activities of spermatozoa undergoing various physiological reactions are already available, such as the use of Ca 2+ for triggering the acrosome reaction in vitro.

Fine structure observations in frozen-etched bovine spermatozoa

A freeze-etching instrument has been constructed using the Mikros VE-20, 4-inch vacuum evaporator as a basic starting point. In the present investigation, this instrument has been employed in the study of bovine spermatozoa frozen by procedures which ensure that a large fraction of the cells remain viable after thawing. Replicas of these frozen cells viewed by electron microscopy show many of the features described by conventional electron microscopic techniques and, in addition, exhibit structures which have not been revealed thus far. These more unique structures include a series of periodic striations associated with membrane in the caudal region of the sperm head, a periodic belt-like band at the base of the head, and a nuclear component consisting of stacked lamellae.

Human sperm head ultrastructure: a freeze-etching study.

The fine structure of human spermatozoa has been explored using freeze-etching, surface-replica, and thin-sectioning methods. The post nuclear sheath region is seen to be a modestly developed structure having few of the elaborate specializations of bull or rabbit spermatozoa. As with other mammalian sperm studied thus far by the freeze-etching or surface-replica methods, the basal portion of the postnuclear sheath terminates in a posterior ring which completely encircles the base of the sperm head. When the low temperature fracturing process exposes acrosomal membrane faces, they are often seen to consist of densely packed arrays of 200 A particles. This is in distinct contrast with plasmalemma faces which possess relatively few, randomly scattered particulates and may be related to enzymatic activities involving the acrosome. Although showing occasional regions of lamellar or linear ordering, human sperm nuclei exposed by cross fracturing appear to be more heterogeneously packed than those of highly flattened bull or rabbit sperm heads. This suggestion is supported by the lack of birefringence exhibited by human spermatozoa.

Media induced alterations of the membrane associated particles of the guinea pig sperm tail

The guinea pig sperm tail was examined by freeze-etching and other techniques to determine changes in structure resulting from in vitro capacitation. Fresh epididymal sperm display profuse strands composed of small particles uniquely associated with the middle piece plasmalemma. Deep-etching shows these strands to be intramembranous structures, whereas a regular double row of particles aligned along the axis of the principle piece is demonstrated as residing on the surface of the cell membrane. The strands of small particles enveloping the middle piece are seen to dissociate during long term incubation in capacitating media and this structural change is correlated with a modification of flagellar pattern as recorded with cine techniques. Acrosomal disruption appears not to be a prerequisite for either the loss of middle piece strands or the change in motility pattern. The possible sequence of events involved in the observed changes as they might relate to the process of capacitation is discussed.

Cell surface changes associated with in vitro capacitation of hamster sperm

The effect of in vitro capacitation of hamster spermatozoa on plasma membrane ultrastructure and surface carbohydrate distribution has been investigated by freeze—fracture and lectin-labeling techniques. The plasma membrane of epididymal sperm bound Con A densely and uniformly over the entire head region, but the plasma membrane of sperm undergoing capacitation developed discrete areas over the acrosome which did not bind Con A. In cells which had undergone the acrosome reaction, the distribution of Con A binding sites over the equatorial segment and postacrosomal region was similar to that of epididymal sperm. Freeze—fracture revealed that some rearrangement of intramembranous particles occurs in the plasma membrane of the equatorial segment of reacted sperm although particle distribution in the postacrosomal region remains unaltered. The achievement of capacitation in these studies was monitored by an in vitro fertilization assay utilizing hamster eggs obtained by superovulation.

A fine-structure study of the Rotifer integument

The fine structure of the hypodermal syncytium and extracellular cuticle of Asplanchna sieboldi show a number of interesting and somewhat unique features. Among these are numerous bulblike invaginations of the outer cell membranes, an unusual dense lamellar band adjacent to the apparently five-layered plasma membrane, and an acid polysaccharide-protein cuticle displaying complex morphology. A tentative working hypothesis is suggested concerning the involvement of various hypodermal organelles and components in cuticle production. Evidence on the nature of the cuticle comes from a number of histochemical tests as well as enzyme digestion and observation of the results with electron microscopy. Adult rotifers are apparently incapable of regenerating cuticle after its enzymatic removal.

Ultrastructural sperm tail defects associated with sperm immotility

Four individuals with a complaint of infertility were evaluated after a semen analysis had demonstrated no motility. The semen analyses were otherwise normal, with the exception of a low count in one subject. All had normal percentages of living sperm. One of the individuals had chronic respiratory disease, and two others had a previous history of genitourinary infection and/or testicular injury. Three subjects possessed antisperm antibodies. Electron microscopy revealed a multiplicity of sperm tail structural defects seen in all specimens. Some of these data support the concept of acquired immotile sperm syndrome(s) with ultrastructural defects, as contrasted with the usual congenital forms of the immotile-cilia syndrome.

Studies on the structure of the postnuclear sheath of water buffalo spermatozoa

Surface replication of chemically treated and sonicated spermatozoa of the Asiatic water buffalo has been utilized to study the relationship of the post-nuclear sheath components to each other and to the plasma membrane. The postnuclear sheath can be considered a complex composed of matrix material, formed elements termed “striations”, and a caudal, posterior ring. These components appear to vary in lability upon being treated by the various reagents and conditions used for disruption. Total removal of postnuclear sheath structures was accomplished after treatment with media containing dithiothreitol. Freeze-etching studies including the use of deep etching conditions support the suggestion that the particulate material comprising the basal striations are in part intramembranous while extending into the postnuclear sheath matrix internally and making contact with (or forming a part of) the surface membrane externally.