Aida Abou-Haila

Mammalian sperm molecules that are potentially important in interaction with female genital tract and egg vestments

Fertilisation is a highly programmed process by which two radically different cells, sperm and egg, unite to form a zygote, a cell with somatic chromosome numbers. Development of the zygote begins immediately after sperm and egg haploid pronuclei come together, pooling their chromosomes to form a single diploid nucleus with the parental genes. Mammalian fertilisation is the net result of a complex set of molecular events which allow the capacitated spermatozoa to recognise and bind to the eggs extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fuse with the egg plasma membrane. Sperm-zona (egg) interaction leading to fertilisation is a species-specific carbohydrate-mediated event which depends on glycan-recognising proteins (glycosyltransferases/glycosidases/lectin-like molecules) on sperm plasma membrane (receptors) and their complementary glycan units (ligands) on ZP. The receptor-ligand interaction event initiates a signal transduction pathway resulting in the exocytosis of acrosomal contents. The hydrolytic action of the sperm glycohydrolases and proteases released at the site of sperm-egg interaction, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of egg investments. This review focuses on sperm molecules believed to be important for the interaction with the female genital tract, passage through cumulus oophorus and attachment to ZP, induction of the acrosome reaction, secondary binding events, and passage through the ZP. An understanding of the expression and modifications of molecules thought to be important in multiple events leading to fertilisation will allow new strategies to block these modifications and alter sperm function.

Evidence for the capacitation-associated membrane priming of mouse spermatozoa

An important feature of male fertility is the physiological priming of mammalian spermatozoa by a multifaceted process referred to as capacitation. It is a prerequisite event before spermatozoa can bind to the eggs extracellular coat, the zona pellucida, and undergo a signal transduction cascade. The net result is the fusion of the plasma membrane (PM) and underlying outer acrosomal membrane at multiple sites and the release of acrosomal contents (i.e., glycohydrolases, proteinases, etc.) at the site of sperm–zona binding. In this study, we have used an indirect immunofluorescence (IIF) assay and other staining approaches to examine capacitation-associated membrane priming of mouse spermatozoa. For IIF studies, we used affinity-purified antibodies against two glycohydrolases that cross-reacted with the acrosomal enzymes only when the uncapacitated spermatozoa were permeabilized. Incubation of spermatozoa in a medium that favors in vitro capacitation induced membrane priming that allowed the antibodies to cross-react with the acrosomal enzymes in capacitating acrosome-intact spermatozoa without permeabilization, as revealed by the appearance of several distinct fluorescent patterns, including an initial immunopositive lining over the acrosome cap to an intense immunopositive reaction throughout the acrosome. These early immunopositive patterns were followed by the appearance of intense fluorescent spots (droplets) that seem to establish contact with the PM in a time-dependent manner. Inclusion of calmodulin, a 17-kDa Ca2+-binding protein which promotes capacitation, in the incubation medium did not alter the overall rate of capacitation; however, its presence accelerated the initial stages of membrane priming. The potential similarities between sperm capacitation and early events of Ca2+-triggered membrane fusion among eukaryotes and among various stations of the secretory and endocytotic pathways are discussed.

Male Contraception: An Overview of the Potential Target Events

The contraceptive options available to men have not changed in several decades and are still limited to the non-surgical methods of the use of a condom, a timely withdrawal, or a surgical procedure that removes a segment of the vas deferens (vasectomy). The first two approaches have relatively higher failure rates whereas the last approach is largely irreversible and may not be suitable for younger men. Thus, providing a safe, effective and readily available contraception for men has remained an unfulfilled goal. In this article, we intend to review the current status of the research and development on male contraceptives. It is apparent that the scientific community in the past few decades has witnessed impressive progress in understanding the basics of male physiology, the knowledge necessary for developing new contraceptive methods for men. We will highlight various new and improved strategies for the regulation of fertility in males. The diverse approaches that are at various stages of development and/or in clinical trials include: 1) administration of hormones, herbal extracts or chemicals to suppress/arrest sperm production in the testes (spermatogenesis); 2) interference with the delivery of spermatozoa during ejaculation by targeted blockage of vas deferens with plugs or chemicals (polymers) that prevent flow of sperm through the vas duct; 3) active or passive immunization of males with well characterized antigens/antibodies which are intended to block sperm function; and 4) administration of site-directed antagonists to block specific sperm function(s) necessary for normal fertilization. All these approaches do not involve surgery and are reversible. Our intention is to discuss the current status of various approaches which show promising results in clinical trials, particularly in China and India, the worlds most populous nations.

Biological Processes that Prepare Mammalian Spermatozoa to Interact with an Egg and Fertilize It

In the mouse and other mammals studied, including man, ejaculated spermatozoa cannot immediately fertilize an egg. They require a certain period of residence in the female genital tract to become functionally competent cells. As spermatozoa traverse through the female genital tract, they undergo multiple biochemical and physiological changes collectively referred to as capacitation. Only capacitated spermatozoa interact with the extracellular egg coat, the zona pellucida. The tight irreversible binding of the opposite gametes triggers a Ca2+-dependent signal transduction cascade. The net result is the fusion of the sperm plasma membrane and the underlying outer acrosomal membrane at multiple sites that causes the release of acrosomal contents at the site of sperm-egg adhesion. The hydrolytic action of the acrosomal enzymes released, along with the hyperactivated beat pattern of the bound spermatozoon, is important factor that directs the sperm to penetrate the egg coat and fertilize the egg. The sperm capacitation and the induction of the acrosomal reaction are Ca2+-dependent signaling events that have been of wide interest to reproductive biologists for over half a century. In this paper, we intend to discuss data from this and other laboratories that highlight the biological processes which prepare spermatozoa to interact with an egg and fertilize it.

Acid glycohydrolases in rat spermatocytes, spermatids and spermatozoa: enzyme activities, biosynthesis and immunolocalization

Mammalian sperm acrosomes contain several glycohydrolases that are thought to aid in the dispersion and digestion of vestments surrounding the egg. In this study, we have used multiple approaches to examine the origin of acrosome-associated glycohdyrdolases. Mixed spermatogenic cells, prepared from rat testis, were separated by unit gravity sedimentation. The purified germ cells (spermatocytes [SC], round spermatids [RS], and elongated/condensed spermatids [E/CS]) contained several glycohydrolase activities. Metabolic labeling in the cell culture, immunoprecipitation, and autoradiographic approaches revealed that β-D-galactosidase was synthesized in SC and RS in 88/90 kDa forms which undergo processing in a cell-specific manner. Immunohistochemical approaches demonstrated that the enzyme was localized in Golgi membranes/vesicles, and lysosome-like structures in SC and RS, and forming/formed acrosome of E/CS.

Identification and androgen regulation of egasyn in the mouse epididymis

The expression and androgen regulation of egasyn, the endoplasmic reticulum-targeting protein of beta-D-glucuronidase, was examined in the mouse-epididymis. The proximal (caput) and distal (corpus & cauda) epididymal tissue extracts were prepared by homogenization and sonication in buffered Triton X-100 solution, and high speed centrifugation. The supernatant when resolved by 2D-PAGE under non-denaturing conditions and stained for esterase activity showed that the distal (but not proximal) epididymis of the normal mouse contain several specific forms of esterases. These forms include a series of four variants (pI 5.2-5.75) with high mobility (HM) and esterase activity, and three faintly staining variants (beginning at pI 6.0) with low mobility (LM). Several lines of evidence indicate that the specific esterases seen in the corpus/cauda epididymidis are egasyn-esterases. Firstly, these molecular forms were not seen in the distal epididymal extracts from the egasyn-deficient mouse. Secondly, the HM forms can be immunoprecipitated with anti-egasyn antibody, suggesting the presence of free egasyn. Finally, the LM forms disappeared after heat treatment (56 degrees C for 8 min), a condition known to dissociate egasyn:beta-D-glucuronidase complex. This result indicates that a small amount of egasyn is complexed with beta-D-glucuronidase. Immunoblotting (Western blot) studies (using anti-egasyn antibody) following resolution of egasyn released from the egasyn:beta-D-glucuronidase complex revealed a single band of an apparent molecular weight 64 kDa in the distal (but not proximal) epididymis, indicating that the mouse epididymal egasyn is identical or very similar to the liver egasyn. Castration of mice lead to the appearance of free and complexed egasyn forms in the proximal epididymis. Testosterone supplementation to the castrated mice resulted in the disappearance of the induced egasyn forms from the caput epididymidis. Taken together, these results indicate that the expression of egasyn in the epididymis is region-specific and is differentially regulated by androgens.

SPERM-EGG INTERACTION AND EXOCYTOSIS OF ACROSOMAL CONTENTS

It is generally accepted that carbohydrate-binding proteins glycosyltransferases/glycosidases/lection-like molecules) on sperm plasma membrane (receptors) recognize and bind to their complementary glycan molecules (ligands) on the egg’s extracellular coat, the zona pellucida (1–3). Thus, sperm-ovum interaction is a carbohydrate-mediated receptor ligand-binding event. This type of binding is analogous to cellular adhesion events of bacteria, veruses, and many pathogens to their respective host cells. Binding of opposite gametes initiates a calcium (Ca2+)- dependent signal transduction cascade resulting in the exocytosis of sperm acrosomal contents (i.e., induction of the acrosome reaction). This step is thought to be a prerequisite event that enables the acrosome-reacted spermatozoa to penetrate the zona pellucida and fertilize the egg.

Importance of Male Fertility Control in Family Planning

The world population, currently estimated to be almost seven billion, is expected to double in less than four decades. The projected population growth will cause severe competition for existing resources, not to mention the issue of overcrowding of the planet and additional greenhouse gases that will have an adverse effect on the ecological health of the planet. A recent survey conducted by the United Nations Population Control Division shows that the majority of todays young men in many countries are willing to participate in family planning by taking full control of their fertility, an important global health issue. However, the contraceptive needs of tens of millions of men/couples go unmet every single day and results in millions of unwanted pregnancies. Ever since the approval of the birth control pill by the Food and Drug Administration (FDA) in 1960, scientists have been hoping for a male equivalent. It has, however, been a difficult road, in part because of the complicated science of the male reproductive system. It is easier to control a monthly event of ovulation in women than to regulate the production of millions of fertile spermatozoa every day in men. Thus, the contraceptive options for men have not changed in decades and are still limited to the use of condoms, a timely withdrawal/pulling out (coitus interruptus) or vasectomy, a minor surgical procedure in which the vas deferens is occluded to prevent the release of spermatozoa during ejaculation. The first two approaches have a relatively higher failure rate, whereas the last approach is largely irreversible and not suitable for younger men. In this article, we will discuss various approaches currently available for men to take control of their fertility. Our intention is to discuss the details of three similar approaches that will provide safe, affordable and reversible contraception for men and are close to being approved for use by millions of men around the globe. The availability of safe, reversible and reliable male contraceptives will allow men and women to take full control of their fertility in family planning.

How close are we in achieving safe affordable and reversible male contraceptives

The world population, currently estimated to be over six billions, is expected to double in the next forty years. The projected growth will cause severe over crowding that will have an adverse effect on the ecological health of the planet. A recent survey by the United Nations found that a majority of men in many countries are willing to participate in family planning by taking full control of their fertility. However, the available contraceptives for men have either higher failure rates or they are irreversible. Thus, the contraceptive needs of tens of millions of men go unmet every day resulting in millions of unwanted pregnancies, and hundreds of thousands of abortions. Since the introduction of oral contraceptive (pill) for women over five decades ago, there have been numerous collaborative efforts by scientists and pharmaceutical companies to improve the effectiveness and delivery of contraceptives to women who wish to safely regulate their reproductive physiology. However, the contraceptive options available to men have not changed in several decades and are still limited to the use of condoms and timely withdrawal (coitus interruptus) or under going a minor surgical procedure (vasectomy) that prevents the release of spermatozoa during ejaculation. The first two methods have relatively higher typical-use failure rates, whereas the last approach is largely irreversible and not suitable for younger men. Despite non-stop efforts worldwide, we may still be several years away from providing safe, effective and affordable male contraceptives which will allow both men and women to participate fully in family planning. In this article, we will discuss various contraceptives currently available to regulate male fertility. In addition, we will summarize potentially new contraceptives for men that are at various stages of research and development. Finally, our intention is to discuss details of two safe, reversible and affordable male contraceptive approaches that are inching closure to being approved for use by the masses in India and China, the worlds two most populous nations.

The Sperm Acrosome: Formation and Contents

The mammalian spermatozoon is a uniquely shaped cell with a head containing the nucleus and enzyme-filled acrosome, and a flagellum (tail) containing contractile apparatus such as the axoneme, cytoskeletal structures and mitochondria. The shape of the sperm head varies from species to species and usually falls into two categories: a sickle shape in rodents and a paddle shape (spatulate) in several larger species including man. This highly specialized cell, capable of delivering the male genome to the egg, is formed in the testes throughout postpubertal male reproductive life span by a regulated process called spermatogenesis. The final phase of this process, referred to as spermiogenesis, is a continuous process beginning with the formation of the round spermatid and concluding with the release of the spermatozoon into the lumen of the seminiferous tubule. The number of programmed steps needed to transform an ordinary looking round spermatid into a hydrodynamically shaped spermatozoon with a fully developed acrosome varies from species to species and ranges from six to eight steps in man to 19 steps in rats and rabbits.