Biology of male fertility control: an overview of various male contraceptive approaches.

The population of our planet continues to grow at an alarming rate. If the growth continues at the present rate, the estimated current world population of about seven billion is expected to double in the next forty years. Accumulated data from surveys by the United Nations Population Control Division suggest that a majority of today's young men in many countries are willing to have fewer children than their parents did. However, the contraceptive options available to them have not changed in several decades. In spite of the general agreement that men, like women, must take full responsibility of their fertility, the availability of safe, reversible and affordable contraceptives for men have lagged behind because of the complexity of the science of the male reproductive system. Thus, the contraceptive needs of millions of men/couples go unmet every single day and results in millions of unwanted pregnancies. In this article, we intend to discuss new hormonal and non-hormonal contraceptive approaches that are at various stages of research and development and may someday provide new contraceptives for men. In addition, we intend to discuss many details of three safe, effective, affordable and reversible vas-based approaches that are inching closer to being approved for use by millions of men in multiple countries. Finally, our intention is to discuss the male contraceptive pill that will soon be available to men only in Indonesia. The availability of these male contraceptives will allow both men and women to take full control of their fertility and participate in slowing down the growth of world population.

Significance of egg's zona pellucida glycoproteins in sperm-egg interaction and fertilization.

Mammalian fertilization is a highly programmed process by which sperm and egg unite to form a zygote, a cell with somatic chromosome numbers. To fertilize an egg, the capacitated (acrosome-intact) spermatozoa recognize and bind to the egg's extracellular glycocalyx coat, the zona pellucida (ZP). The tight and irreversible binding of the opposite gametes in the mouse and many other species studied, including man, results in the opening of Ca2+ channels on sperm plasma membrane (PM) and influx of Ca2+. The transient rise in Ca2+ and other second messengers, such as cAMP and IP3, initiates a cascade of signaling events that elevate sperm pH and triggers the fusion of the sperm PM and underlying outer acrosomal membrane at multiple sites (induction of the acrosomal reaction). The fusion of the two membranes results in the exocytosis of acrosomal contents at the site of sperm-egg adhesion. The hydrolytic action of the acrosomal enzymes (glycohydrolases, proteinases, esterases, sulfatases etc), released at the site of sperm-egg adhesion, along with the enhanced thrust generated by the hyperactivated spermatozoon, are important factors that regulate the penetration of the ZP and the fusion of the acrosome-reacted spermatozoon with the egg. Evidence accumulated over the past two decades strongly suggests that glycan units of the ZP have a significant role in the recognition and adhesion of the opposite gametes and induction of the AR. In this review article, we intend to highlight well programmed molecular events that results in the sperm-egg adhesion and fertilization. Our intention is also to discuss the increasing controversy on the role of ZP glycan chains in sperm-egg interactions.

Molecular events that regulate mammalian fertilization.

Mammalian fertilization is the net result of a highly programmed sequence of molecular events that collectively result in the union of two radically different looking haploid cells, sperm and egg, to form a diploid zygote. For successful fertilization, sperm cells undergo continuous modifications during their formation in the testis, maturation in the epididymis, and capacitation in the female genital tract. Only capacitated acrosome-intact spermatozoa are capable of binding to the egg's extracellular coat, the zona pellucida (ZP) in a receptor-ligand manner. The species-specific irreversible binding of the opposite gametes elevates intrasperm Ca2+ and triggers a signal transduction cascade that results in the fusion of the sperm plasma membrane and outer acrosomal membrane at multiple sites (i.e., induction of the acrosomal reaction) and the secretion of acrosomal contents. The hydrolytic action of the acrosomal enzymes (i.e., glycohydrolases, proteinases etc.) released at the site of sperm-egg binding along with the hyperactivated beat pattern of the bound spermatozoon, are important factors that regulate its penetration of the ZP and fertilization of the egg. In this article, we intend to discuss data from this and other laboratories that provide useful insights into biology underlying sperm development in the testis, maturation in the epididymis, capacitation in the female genital tract, sperm-egg interaction, and induction of the acrosome reaction (AR) before the acrosome reacted sperm can fertilize an egg. Our intention is also to discuss how Ca2+ signaling cascades regulate sperm functions and male fertility. Finally, we will discuss sperm molecules that are under intensive research to regulate male fertility.

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 egg's 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.

Rat spermatogenic cell beta-D-galactosidase: characterization, biosynthesis, and immunolocalization.

In this study we have demonstrated that the rat sperm acrosomal beta-d-galactosidase is expressed in late spermatocytes and spermatids (round, elongated/condensed) during spermatogenesis. The enzyme is an exoglycohydrolase which, along with other exoglycohydrolases and proteases, is thought to aid in penetration of the zona pellucida, the extracellular glycocalyx that surrounds the mammalian egg. The presence of the enzyme in spermatocytes was confirmed by multiple approaches using biochemical, biosynthetic, and immunohistochemical protocols. The germ cells (spermatocytes, round spermatids, and elongated/condensed spermatids), purified from rat testis, were found to contain beta-galactosidase and four other glycohydrolases (beta-d-glucuronidase, alpha-d-mannosidase, alpha-l-fucosidase, and beta-N-acetylglucosaminidase). With the exception of alpha-l-fucosidase, the other enzymes assayed demonstrated a two- to threefold higher activity per cell in spermatocytes than in round spermatids. Immunoblotting approaches of affinity-purified germ cell extracts demonstrated several molecular forms of beta-galactosidase in spermatocytes and round spermatids; one of these forms (62 kDa) was seen only in round spermatids. The biosynthetic approach demonstrated that the enzyme is synthesized in spermatocytes and round spermatids in culture in high-molecular-weight precursor forms (90/88 kDa) which undergo processing to lower molecular weight mature forms in a cell-specific manner. The net result is the formation of predominantly 64- and 62-kDa forms in spermatocytes and round spermatids, respectively. The conversion of precursor forms to mature forms in the diploid and haploid cells in culture is rapid with t(1/2) of 6.5 and 9.0 h, respectively. Immunohistochemical approaches revealed an immunopositive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosome-like structures in the late spermatocytes and early round spermatids. The forming/formed acrosome in round and elongated spermatids was also immunoreactive.

Mammalian sperm acrosome: formation, contents, and function.

Sperm-egg interaction is a carbohydrate-mediated species-specific event which initiates a signal transduction cascade resulting in the exocytosis of sperm acrosomal contents (i.e., the acrosome reaction). This step is believed to be a prerequisite which enables the acrosome-reacted spermatozoa to penetrate the zona pellucida (ZP) and fertilize the egg. Successful fertilization in the mouse and several other species, including man, involves several sequential steps. These are (1) sperm capacitation in the female genital tract; (2) binding of capacitated spermatozoa to the egg's extracellular coat, the ZP; (3) induction of acrosome reaction (i.e., sperm activation); (4) penetration of the ZP; and (5) fusion of spermatozoon with the egg vitelline membrane. This minireview focuses on the most important aspects of the sperm acrosome, from its formation during sperm development in the testis (spermatogenesis) to its modification in the epididymis and function following sperm-egg interaction. Special emphasis has been given to spermatogenesis, a complex process involving multiple molecular events during mitotic cell division, meiosis, and the process of spermiogenesis. The last event is the final phase when a nondividing round spermatid is transformed into the complex structure of the spermatozoon containing a well-developed acrosome. Our intention is also to briefly discuss the functional significance of the contents of the sperm acrosome during fertilization. It is important to mention that only the carbohydrate-recognizing receptor molecules (glycohydrolases, glycosyltransferases, and/or lectin-like molecules) present on the surface of capacitated spermatozoa are capable of binding to their complementary glycan chains on the ZP. The species-specific binding event starts a calcium-dependent signal transduction pathway resulting in sperm activation. The hydrolytic and proteolytic enzymes released at the site of sperm-zona interaction along with the enhanced thrust of the hyperactivated beat pattern of the bound spermatozoon, are important factors in regulating the penetration of the zona-intact egg.

Characterization and immunolocalization of beta-D-glucuronidase in mouse testicular germ cells and spermatozoa.

Spermatozoa released from the seminiferous tubules are terminally differentiated cells with no known synthetic activity. Their components are synthesized in the spermatogenic cells during spermatogenesis. In this study, we report the characterization and immunolocalization of beta-glucuronidase in mouse testicular germ cells and spermatozoa. The enzyme is an exoglycohydrolase with dual localization, being present in lysosomes and endoplasmic reticulum of several mouse and rat tissues. The purified germ cell preparations (spermatocytes, round spermatids, and condensed/elongated spermatids) when assayed for beta-glucuronidase activity showed that the spermatocytes contained five times more enzyme activity per cell than the spermatids. Polyacrylamide gel electrophoresis, carried out under native and denaturing conditions, demonstrated that the germ cells express only the lysosomal form of the enzyme (pI 5.5-6.0) with a subunit molecular mass of 74 kDa. Immunocytochemical studies revealed a positive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosomes of late spermatocytes (pachytene spermatocytes) and a stage-specific localization during spermiogenesis. The forming or formed acrosome of the elongated spermatids (stages 9-16) and epididymal spermatozoa was highly immunopositive. Comparison of immunoprecipitation curves and kinetic properties of the enzyme present in spermatocytes and spermatozoa revealed no major differences. Taken together, our results demonstrate that beta-glucuronidase activities present in the lysosomes of spermatocytes and the sperm acrosome are kinetically and immunologically similar.

Rat sperm surface mannosidase is first expressed on the plasma membrane of testicular germ cells.

In previous publications (Tulsiani et al., Biochem J 1993; 290:427-436 and Tulsiani et al., Dev Biol 1995; 167:584-595), we reported that sperm surface mannosidase is present in rat testis and is modified during spermatogenesis and sperm maturation. The present studies were directed towards examining the origin of alpha-D-mannosidase activity present on fertile spermatozoa. Mixed germ cells prepared after sequential enzymatic digestions of rat testis were separated by unit gravity sedimentation using 2-4% linear bovine serum albumin gradient. Fractions enriched in spermatocytes, round spermatids, and condensed/elongated spermatids (> 95% pure cells) were separately pooled and assayed for [3H]Man9-mannosidase activity before (intact) and after lysis with Triton X-100. Interestingly, the cells contained a significant level of alpha-D-mannosidase activity. Approximately 70% of the total [3H]Man9-mannosidase activity present in the detergent-solubilized germ cell extract cross-reacted with anti-rat sperm mannosidase, and 25% of the activity cross-reacted with anti-Golgi mannosidase I. This result indicates that most of the mannosidase activity present in the germ cell extract is antigenically similar to the enzyme present on the cauda spermatozoa. Using cell fractionation techniques, we obtained evidence suggesting that the germ cell-associated mannosidase activity is an integral component of the plasma membranes. Taken together, these results indicate that sperm surface mannosidase is first expressed on the testicular germ cells.

The biological and functional significance of the sperm acrosome and acrosomal enzymes in mammalian fertilization.

The mammalian spermatozoon undergoes continuous modifications during spermatogenesis, maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are capable of binding the zona-intact egg and undergoing the acrosome reaction. The fertilization process is a net result of multiple molecular events which enable ejaculated spermatozoa to recognize and bind to the egg's extracellular coat, the zona pellucida (ZP). Sperm-egg interaction is a species-specific event which is initiated by the recognition and binding of complementary molecule(s) present on sperm plasma membrane (receptor) and the surface of the ZP (ligand). This is a carbohydrate-mediated event which initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be a prerequisite which enables the acrosome reacted spermatozoa to penetrate the ZP and fertilize the egg. This review focuses on the formation and contents of the sperm acrosome as well as the mechanisms underlying the induction of the acrosome reaction. Special emphasis has been laid on the synthesis, processing, substrate specificity, and mechanism of action of the acid glycohydrolases present within the acrosome. The hydrolytic action of glycohydrolases and proteases released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of ZP. We have discussed the most recent studies which have attempted to explain signal transduction pathways leading to the acrosomal exocytosis.

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.

Characterization of glycoconjugates in the epididymal epithelium and luminal fluid during postnatal development of the mouse.

The qualitative nature and distribution of glycoproteins in the mouse epididymis during postnatal development was examined by using lectin cytochemical procedures on paraffin sections and lectin blots on electrophoretically separated luminal fluid polypeptides transferred onto nitrocellulose. Histochemical results revealed the presence of glycoproteins with terminal alpha-D-mannose, N-acetyl-D-glucosamine and neuraminic acid in the principal cells along the epididymis during early stages of development (1st week), and glycoproteins containing terminal alpha-L-fucose, N-acetyl-D-galactosamine and alpha-D-galactose in specific regions of the duct during the differentiation state (2nd-3rd week). Lectin staining localized in the Golgi region and at the apical surface increased during development. Specific changes occurred with age and between cell types. Examination of the epididymal luminal fluid glycopeptides by lectin blot analysis revealed the presence of a large number of glycoproteins with various saccharide moieties at 7 days of age. Epididymal differentiation was accompanied either by the disappearance of some glycoproteins (apparent molecular mass: 16, 17.5, 22, 28, 30 and 74 kDa) or the appearance of new glycoproteins in the proximal (23, 13 kDa) and distal regions (29, 20.5, 19 and 14.4 kDa), or along the entire epididymal duct (26 kDa). The main changes occurred in the epididymis of 21-day-old mice and were completed before spermatozoa reached the epididymal lumen.

Enzyme histochemical characteristics of osteoblasts and mononucleated osteoclasts in a teleost fish with acellular bone (Oreochromis niloticus, Cichlidae).

Bone resorption by mononucleated cells was studied in the acellular bone of a teleost fish (Oreochromis niloticus) by histological and enzyme histochemical observations and by transmission electron microscopy. Bone resorbing cells (osteoclasts) were identified by their location at the sites of bone resorption, their frequent association with a band of concentrated activity of tartrate-resistant acid phosphatase at the bone surface and by the presence or lack of certain enzymes. Tartrate-resistant acid phosphatase was used as a marker for osteoclasts, and alkaline phosphatase as a marker for osteoblasts. Osteoclasts in O. niloticus are not multinucleated; however, during intense bone resorption, they form cell aggregations that resemble multinucleated giant cells in mammals. Conversely, during less intense bone degradation, osteoclasts are flat, have long narrow cytoplasmic processes and resemble the bone-lining cells of mammals. All bone-resorbing cells in O. niloticus are mononucleated and lack a ruffled border. Similarities to and differences from bone resorption by mononucleated cells in mammals are discussed.

Androgen regulation of molecular forms of beta-D-glucuronidase in the mouse epididymis: comparison with liver and kidney.

The expression and androgen regulation of beta-glucuronidase molecular forms were examined in mouse epididymis, liver, and kidney. Two-dimensional polyacrylamide gel electrophoresis performed under nondenaturing conditions showed that, compared to liver and kidney, which contain four microsomal (M1-M4) and a major lysosomal (L) form of beta-glucuronidase, the epididymis revealed regional differences and tissue specificity in the expression of the various molecular forms of the enzyme. Only the lysosomal form (pI 5.4-6.1) is present in the caput epididymidis while the corpus/cauda contains the lysosomal form, the free X form (pI 5.9-6.3) and the four microsomal forms (X form complexed with egasyn). Mutant mice that lack egasyn have no microsomal forms in the distal epididymis. In epididymal fluid, the lysosomal form is found throughout the epididymis, whereas the X form appears only in the corpus/cauda epididymidis. Sodium dodecyl Sulfate (SDS)-gel electrophoresis and western blot analysis of immunoprecipitated beta-glucuronidase revealed only one band corresponding to the L form (apparent molecular weight 74 kDa) in the caput epididymidis and two bands in the corpus/cauda (apparent molecular weights 73 and 75 kDa), corresponding to L and X forms, respectively. Castration of mice led to the suppression of the regional differences in the appearance of X and M forms in the epididymis. Testosterone supplementation to castrated mice restored the characteristic electrophoretic pattern of beta-glucuronidase in the caput epididymidis. In the liver and kidney, castration has no effect on the expression of the molecular forms, whereas androgen treatment induced the X form in the kidney. Histochemical localization of beta-glucuronidase confirmed the region specificity seen in the epididymis and in addition revealed cell specificity in the expression of beta-glucuronidase. These results indicate that beta-glucuronidase shows tissue specificity and, in the case of the epididymis, region and cell specificity. In addition, the enzyme in the different tissues responds differentially to androgens.

Synthesis, characterization and hormonal regulation of epididymal proteins during postnatal development of the mouse.

Maturation of mammalian spermatozoa depends on their interactions with epididymal proteins. The incorporation of 35S-methionine into these proteins was investigated by in vitro incubation of tissue minces from the mouse epididymis at different ages of postnatal development. The greatest amount of incorporation per wet weight of tissue was seen in 7 to 21-day-old mice. It decreased progressively during development while the rate of proteins released into the medium remained almost constant until the adult state. Separation of labeled proteins on sodium dodecyl sulphate polyacrylamide gels followed by fluorography showed that the great majority of secretory proteins synthesized in adult mouse epididymis could be recovered already from 7-day-old animals. Regional differences appeared at 21 days of age. These were marked by the secretion of proteins characteristic of the proximal (26, 25, 20, 19 kDa) and distal (44, 29 kDa) epididymis. Analysis of cytosol and luminal fluid proteins from prepubertal and adult epididymis revealed a number of proteins of the same mobility as those synthesized and secreted in vitro. Among the luminal proteins which showed variations during development and regional differences, four (29, 26, 20, 19 kDa) were characteristics of the epididymis and three (88, 34, 13 kDa) comigrated with testicular components. Castration or estrogen treatment of prepubertal mice for 4, 3 and 2 weeks inhibited or reduced the synthesis of the luminal proteins which appeared during postnatal development and/or presented regional differences. Testosterone replacement of castrated mice reversed this effect and induced the secretion of new proteins (37, 24 kDa).(ABSTRACT TRUNCATED AT 250 WORDS)

Selective action of androgens on the molecular forms of esterases characterized by two-dimensional gel electrophoresis in the epididymis and vas deferens of the mouse.

Molecular forms of esterases were resolved in non-denaturing conditions by using two-dimensional gel electrophoresis with isoelectric focusing in the first dimension and a time-dependent polyacrylamide gradient gel electrophoresis (PAGGE) in the second dimension. This procedure was used to analyse sequential changes in esterase composition along the excurrent genital duct of the mouse and to initiate a specific identification of the androgen-regulated molecular forms. Almost all the 68 variants (pH 3.9-6.4 and 50-300 kDa) revealed by alpha-naphtyl acetate from the fluids of the three parts of the epididymis (caput, corpus, cauda) and vas deferens, could be assigned to the carboxylesterase group as shown by their action on various substrates and sensitivity to inhibitors. Some of these variants co-migrated with those in the serum and testis, whereas other enzyme forms made their first appearance in the caput (13), in the corpus (26) and in the vas deferens (3). The major changes occurred between the caput and the corpus of the epididymis. Only a few acidic spots were not revealed after neuraminidase digestion. Castration of mice (4 weeks) resulted in inhibition of the activity of 34 esterase forms, and thus abolished most of the regional differences in the excurrent duct system. By re-initiating or repressing the synthesis of regional esterase variants, testosterone supplementation (2 and/or 4 weeks) of castrated animals restored the normal esterase pattern in the three epididymal parts, but not in the vas deferens. The major effect of efferent duct ligation (4 weeks) was the emergence in the corpus and cauda of the epididymis of two variants found in the caput of uncastrated mice.

Micro two-dimensional gel electrophoresis of serum and testis esterases from different strains of mice.

Two-dimensional electrophoresis with time-dependent polyacrylamide gradient gel electrophoresis (PAGGE) in the second dimension was applied to the separation of native molecular forms of esterases from serum and testis of four strains of mice (C57BL/6J, Swiss OF1, F1 hybrid derived from these two populations and Tfm). In Phast System, a modified pH 3-9 gradient, a linear 8-25% gel gradient and a migration time corresponding to 300 Vh, were found to provide the best conditions for esterase analysis. About 70 esterase-active fractions could be separated with good reproducibility. The variants were characterized by their pI (3.9-7.35), their relative mobility and the visual estimation of their susceptibility towards neuraminidase and different esterase inhibitors. In the two tissues, the distribution of the esterase variants corresponded to a 50-500 kDa molecular mass range of calibration proteins, but most of the serum and testis-specific isoforms were confined to the 59-72 kDa range. All serum variants contained a terminal N-acetylneuraminic acid residue, whereas only the testicular esterases in common with those in serum appeared sensitive to neuraminidase. Cholinesterases with a low relative mobility and carboxylesterases with a high relative mobility were detected in serum, while carboxylesterases accounted for the greatest part in the testis which also contained cholinesterases and acetylesterases. Minor interspecies differences were found between C57BL/6J and Swiss OF1 esterases. The expression of two variants which differed between these two species seemed intermediate for the hybrid originating from these two populations. Two new spots were detected in the two-dimensional map of esterases from the strain bearing the Tfm mutation.

Organization of the internal membrane system in the principal cells of the mouse epididymis after osmium impregnation.

The ultrastructure of the principal cells of the mouse epididymis was studied using osmium impregnation techniques which have the advantage that the endoplasmic reticulum (ER) content displays a positive reactivity after glutaraldehyde fixation whereas the Golgi condensing vacuoles are negative. In the proximal part (caput) of the epididymis, the Golgi apparatus formed a large supranuclear area filled with electronluscent secretory vacuoles while, in the medial (corpus) and distal (cauda) parts, dictyosomes were small and sparse with few secretory vacuoles. In all the principal cells of the caput, the supranuclear ER cisternae were heterogeneously impregnated. In the corpus and cauda, the ER appeared as an extensive continuous network of canaliculi and saccules which were fenestrated when surrounding mitochondria. The ER content was homogeneously stained but impregnation intensity varied from cell to cell. In the apex of the caput cells, numerous impregnated or electronluscent vesicles were seen in close apposition to the plasma membrane, while in the corpus and cauda some Golgi vacuoles and extensions of ER canaliculi were observed in the terminal webb region. Thus, in the epididymal caput, osmium impregnation suggested that two distinct secretory pathways were functioning continuously. The first corresponded to the transport of proteins to the cell membrane by the Golgi condensing vacuoles. The second might only affect the small impregnated vesicles of the ER, through which proteins bypassed the Golgi apparatus and were exported towards the lumen. In the corpus and cauda, the network organization of the ER and the association of fenestrated cisternae with mitochondria (also found in absorptive epithelial cells) supported the view of a predominant absorptive function in these epididymal parts.

Postnatal differentiation and endocrine control of esterase isoenzymes in the mouse epididymis.

At 2 weeks of age, 11 isoenzymes were expressed and similar banding patterns on vertical polyacrylamide gel electrophoresis (PAGE), stained by alpha- or beta-naphthyl acetate as a substrate, were obtained for tissues or fluids from the proximal and the distal parts of the mouse epididymis. After this period, the emergence of new bands or the disappearance of certain others led to a regional differentiation which appeared progressively in tissues and fluids, earlier in the distal part than in the proximal part. The changes occurring during epididymal differentiation affected the isoenzymes specific to the epididymis more than those common to testis and serum. Castration of adult mice induced a decrease in esterase activity and changes in the number of isoenzymes, leading to the loss of regional specificity of the banding patterns. The dedifferentiation process modified the electrophoretic profiles of the distal part only. Androgen replacement restored the regional specificity of cytosol banding patterns after 2 weeks of treatment and the normal intensity of bands after 4 weeks. Some differences in the fluid isoenzymes nevertheless persisted. The androgen-dependence of esterase isoenzymes can be attributed to circulatory hormones rather than to androgens from the testis via the rete testis as shown by efferent ductule ligation which did not modify the epididymal esterase profiles.