Characterization of hormonally regulated secretory proteins from the caput epididymidis of the rabbit.

1. The incorporation of [35S]methionine into protein was investigated in tissue minces from different regions of the rabbit epididymis incubated in vitro. Rates of synthesis were in the order: epididymal regions 2-5 greater than region 7 greater than region 6 greater than region 1 greater than region 8 greater than ductus deferens greater than ductuli efferentes. 2. Separation of labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate followed by fluorography revealed that one protein (mol.wt. approx. 90 000) was characteristic of region 1, four proteins (one of mol.wt. 54 000 and three of mol.wt. 20 000) were synthesized principally in regions 2-5, and one protein (mol.wt. 22 500) was produced mainly in regions 6, 7 and 8. 3. Castration for 14 days decreased incorporation of [35S]methionine into total protein to less than 10% of that in controls in all regions of the epididymis. However, testosterone treatment for a further period of 14 days restored protein synthesis to normal values in regions 6, 7 and 8, but not in region 1 or regions 2-5. In regions 2-5 the synthesis of three proteins of mol.wt. 20 000 declined after castration, but was not stimulated by exogenous testosterone. Since the 20 000-mol.wt. proteins were major tissue proteins, accounting for 16-25% of the total synthesized, they were used as markers for investigating hormone action in the epididymis. 4. Castration followed immediately by testosterone treatment or ligation of the ductuli efferentes resulted in a decrease in their synthesis, suggesting that they are partially dependent on factors in testicular fluid. Purification and characterization showed them to be acidic glycoproteins with a number of biochemical and immunological properties in common. 5. It is suggested that there is a synergistic action between blood androgens and factors in testicular fluid in regulating protein synthesis in the proximal regions of the rabbit epididymis.     

Changes in the protein composition of rat spermatozoa during maturation in the epididymis

Spermatozoa from the testis and cauda epididymidis were solubilized by detergent treatment and electrophoresis on SDS polyacrylamide gels revealed that the relative amounts of 13 detergent-extractable proteins decreased during passage of spermatozoa through the epididymis, 6 increased, whilst the remainder showed little or no change. Lactoperoxidase-catalysed iodination of plasma membrane proteins showed that the components carrying most of the label in testicular spermatozoa had Mr values of 110 000, 94 000, 84 000, 55 000 and 42 000 whereas on cauda epididymal spermatozoa the Mr values were 47 000, 24 000, 17 000, 14 500 and 13 500. Substantial differences were also noted in the protein composition of rete testis fluid and cauda epididymal plasma. The results support the concept that there is a considerable reorganization of the molecular architecture of the plasma membrane of spermatozoa during maturation in the epididymis.     

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [¹⁴C]leucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3°C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of [¹⁴C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of [¹⁴C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.     

Two-dimensional electrophoresis of proteins in principal cells, spermatozoa, and fluid associated with the rat epididymis

Spermatozoa, fluids, and principal cells from different regions of the epididymis were characterized by two-dimensional electrophoresis. Rete testis fluid was collected after 36-h efferent duct ligation, and cauda epididymal fluid was collected by retrograde perfusion through the vas deferens. Spermatozoa were collected after their exudation from minced caput and corpus epididymal tissue. Principal cells were recovered after enzymatic disaggregation and centrifugal elutriation of epididymides. Two-dimensional polyacrylamide gel electrophoresis was used to prepare protein profiles of all samples. Comparison of the proteins found in rete testis fluid versus those found in cauda epididymal fluid revealed a dramatic change in composition, including the loss, addition, or retention of specific proteins as well as changes in the relative concentrations of certain proteins. Prominent cauda epididymal fluid proteins, possibly contributed by the epididymal epithelium, were detected at 16, 23, and 34 kDa. After epididymal transit, a considerable decrease was observed in the number of aqueous-soluble sperm proteins. Differences in the protein composition of epididymal epithelial principal cells from the caput versus corpus epididymidis were also noted, suggesting that functional differences exist for these epididymal regions. Of particular interest was the occurrence of a prominent protein of approximately 20-23 kDa found in all sperm samples, in fluids, and in caput and corpus principal cells. However, this protein was absent in cauda epididymal sperm after 36-h efferent duct ligation. The rapid loss of this protein from sperm after efferent duct ligation suggests that this surgical intervention may affect spermatozoa residing within the epididymis.     

Immunocytochemical localization of the major glycoprotein of epididymal fluid from the cauda in the epithelium of the mouse epididymis

Summary The most abundant protein in fluid from the mouse cauda epididymidis, designated CP 27, is a glycoprotein that migrates at approximately 27000 daltons on SDS-polyacrylamide gels. Samples of CP 27 were isolated by preparative gel electrophoresis and were used to raise a guinea-pig polyclonal antiserum, which reacted with a single band on western blots of caudal epididymal fluid. This antiserum was used for immunocytochemical localization of CP 27 in histological sections of mouse epididymis using the peroxidase-antiperoxidase and protein A-gold methods. The most proximal staining with anti-CP 27 was in segment 6 of the distal caput epididymidis, where the lumen and a portion of the supranuclear cytoplasm of principal cells were stained. In contrast, in the distal corpus and cauda epididymidis (segments 8–11), there was pronounced staining of the luminal contents, stereocilia, and scattered cells identified as the light cells of the epididymal epithelium. Although CP 27 was found in the epididymal lumen of all segments distal to segment 6, the intensity of staining appeared to decline distally in the cauda epididymidis. Control sections exposed to pre-immune serum instead of anti-CP 27 showed no reaction. The results suggest that CP 27, the major glycoprotein of cauda epididymal fluid, is synthesized by principal cells of segment 6 of the distal caput epididymidis. CP 27 may be among the substances absorbed from the lumen by the light cells of the distal epididymis.     

Epididymal lipocalin-type prostaglandin D2 synthase: identification using mass spectrometry, messenger RNA localization, and immunodetection in mouse, rat, hamster, and monkey.

This study identified prostaglandin D2 synthase (PGDS) in murine epididymal fluid using a proteomic approach combining two-dimensional (2D) gel electrophoresis and mass spectrometry (MS). The caudal epididymal fluid was collected by retroperfusion, and proteins were separated by 2D gel electrophoresis followed by matrix-assisted laser desorption ionization MS analyses after trypsin digestion. The identification was based on the protein-specific peptide map as well as on sequence information generated by nano-electrospray ionization MS/MS. By in situ hybridization, the mRNA was detected in caput, corpus, and cauda, but it was not detected in the initial segment. The PGDS protein was mostly detected in the corpus and cauda by Western blot analysis and immunohistochemistry using a specific polyclonal antibody. In caudal fluid, PGDS was distributed among several isoforms (pI range, 6.5-8.8), suggesting that this protein undergoes posttranslational modification of its primary sequence. After N-glycanase digestion, the molecular mass decreased from 20-25 to 18.5 kDa, its theoretical mass. The PGDS was also detected in the epididymis of rat, hamster, and cynomolgus monkey from the caput to the cauda. In conclusion, MS is a powerful and accurate technique that allows unambiguous identification of the murine epididymal PGDS. The protein is 1) present throughout the epididymis, except in the initial segment, with an increasing luminal concentration from distal caput to cauda; 2) a major protein in caudal fluid; 3) an N-glycosylated, highly polymorphic protein; and 4) conserved during evolution.     

Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat

Sulfated glycoprotein-2 (SGP-2) is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. SGP-2 mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications. The detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. SGP-2 was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins.     

Short-term zinc deficiency in diet induces increased oxidative stress in testes and epididymis of rats

In order to determine the effects of Zinc deficient diet on oxidative stress in testis and epididymis, various parameters viz: total proteins, lipid peroxidation, hydroperoxides, antioxidant capacity and enzymatic activities are evaluated in rats fed on zinc deficient diet for 2, 4 and 6 weeks. Total proteins, water and lipid solouble antioxidant capacity decreased while lipid peroxidation (TBARS) and hydroperoxides concentration increased in testes, caput and cauda epididymis except in 2ZD (testes) where hydroperoxides revealed a significant decrease. GSH decreased in testes and caput and cauda epididymis. GPx and gamma-GT activities increased in testes and caput and cauda epididymis of zinc deficient rats. Further, GST increased in testes but exhibited decreases after 2 and 4 weeks and an increase after 6 weeks in caput and cauda epididymis of zinc deficient rats. GR activities decreased in testes but it increased in caput and cauda epididymis of zinc deficient rats. Thus, zinc deprivation results in increased sensitivity to oxidative stress. All these may have been as a consequence of increased ROS generation and/or decreased zinc dependent antioxidant processes.     

Inhibition of protein synthesis in vitro by proteins from the seeds of Momordica charantia (bitter pear melon).

1. A haemagglutinating lectin was purified from the seeds of Momordica charantia by affinity chromatography on Sepharose 4B and on acid-treated Sepharose 6B. It has mol.wt. 115 000 and consists of four subunits, of mol.wts. 30 500, 29 000, 28 500 and 27 000. 2. The lectin inhibits protein synthesis by a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of approx. 5 micrograms/ml. Protein synthesis by Yoshida ascites cells is partially inhibited by the lectin at a concentration of 100 micrograms/ml. 3. From the same seeds another protein was purified which has mol.wt. 23 000 and is a very potent inhibitor of protein synthesis in the lysate system, with an ID50 of 1.8 ng/ml. This inhibitor has no effect on protein synthesis by Yoshida cells, and has no haemagglutinating properties. 4. Artemia salina ribosomes preincubated with the lectin or with the inhibitor lose their capacity to perform protein synthesis. The proteins seem to act catalytically, since they inactivate a molar excess of ribosomes. 5. The lectin and the inhibitor are somewhat toxic to mice, the LD50 being 316 and 340 micrograms/100 g body wt. respectively.     

Characterization of a 23 kDa sperm-binding polypeptide of the golden hamster epididymis

A 23 kDa polypeptide has been identified on the flagellum of sperm obtained from the cauda epididymis of the golden hamster. A monospecific antiserum to the 23 kDa hamster polypeptide was prepared and used to study its distribution on sperm, in the epididymis, and in epididymal fluid. In the cauda, the polypeptide is found on the midpiece and endpiece of the sperm tail, in detergent extracts of sperm, and in epididymal luminal fluid-enriched fractions. It is not present on sperm or in luminal fluid-enriched fractions from the caput epididymis. Immunocytochemical staining of epididymal tissue has demonstrated the 23 kDa polypeptide in the Golgi region of the principal cells of the proximal cauda and on sperm in the tubules of this segment and in tubules distal to it. Antiserum to the 23 kDa golden hamster polypeptide cross-reacts with sperm from rats and Chinese hamsters, but not with sperm from rabbits, cattle, mice, and guinea pigs. The antigen is localized to the tail of sperm obtained from the cauda of the rat and from the distal caput of the Chinese hamster. Immunoblots of detergent extracts of sperm and luminal fluid-enriched fractions from these two species reveal a 26 dKa polypeptide that is immunologically related to the golden hamster polypeptide.     

Molecular weights and metabolism of rat brain proteins

1. Rats were injected with [U-¹⁴C]glucose and after various intervals extracts of whole brain proteins (and in some cases proteins from liver, blood and heart) were prepared by high-speed centrifugation of homogenates in 0.9% sodium chloride or 0.5% sodium deoxycholate. 2. The extracts were subjected to gel filtration on columns of Sephadex G-200 equilibrated with 0.9% sodium chloride or 0.5% sodium deoxycholate. 3. Extracts prepared with both solvents displayed on gel filtration a continuous range of proteins of approximate molecular weights ranging from less than 2×10⁴ to more than 8×10⁵. 4. The relative amount of the large proteins (mol.wt.>8×10⁵) was conspicuously higher in brain and liver than in blood. 5. At 15min after the injection of [U-¹⁴C]glucose the smaller protein molecules (mol.wt.<2×10⁴) were significantly radioactive, whereas no ¹⁴C could be detected in the larger (mol.wt.>2×10⁴) protein molecules. The labelling of all protein samples was similar within 4h after injection of [U-¹⁴C]glucose. Fractionation of brain proteins into distinctly different groups by the methods used in the present work yielded protein samples with a specific radioactivity comparable with that of total brain protein. 6. No evidence could be obtained by the methods used in the present and previous work to indicate the presence of a significant amount of `metabolically inert protein' in the brain. 7. It is concluded that: (a) most or all of the brain proteins are in a dynamic state of equilibrium between continuous catabolism and anabolism; (b) the continuous conversion of glucose into protein is an important part of the maintenance of this equilibrium and of the homoeostasis of brain proteins in vivo.     

Tissue and cell specificity of immobilin biosynthesis

The mechanisms for the initiation of sperm motility have been poorly understood until recently. Immobilin is a novel mucin glycoprotein of high molecular weight found in the cauda epididymis of the rat that, at concentrations equivalent to those found in native cauda epididymal fluid, reversibly inhibits sperm motility. In this study, immobilin was purified from rat cauda epididymal fluid to apparent homogeneity and used to generate polyclonal antibody in rabbits. The antibody was characterized by immunoblotting, and immunofluorescence was used to localize immobilin in paraffin sections of components of the reproductive system of adult male rats. Immobilin was not detectable in the efferent duct and was first detectable in the apical portion of some epithelial cells of the initial segment of the caput epididymis. Immobilin was detectable intracellularly only in cells of the caput epididymis. In the corpus and cauda epididymis immobilin was detectable only in the lumen of the tubules. Immunoprecipitation of immobilin radiolabeled in vitro confirmed that immobilin biosynthesis in the adult rat is restricted to the caput epididymis. Principal cells in the caput epididymis synthesize immobilin and secrete it into the lumen of the tubules to travel with the sperm into the cauda.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

A maturation-related differential phosphorylation of the plasma membrane proteins of the epididymal spermatozoa of the hamster by endogenous protein kinases

When the plasma membranes of caput and cauda epididymal spermatozoa of hamster were evaluated for their ability to undergo phosphorylation, a differential phosphorylation of the membrane proteins was observed. In the plasma membranes of the caput epididymal spermatozoa (immature), twelve proteins were phosphorylated (100, 76, 67, 65, 55, 52, 47, 42, 38, 32, 30, and 20 kD), whereas in the plasma membranes of cauda epididymal spermatozoa (mature), a differential phosphorylation pattern was observed with respect to the 94, 67, 52, and 47 kD proteins. The 94 kD protein was found to be phosphorylated and the 67 kD protein was found to be not phosphorylated in cauda spermatozoal plasma membrane (Cd SPM) in contrast to this protein in caput spermatozoal plasma membrane (Cpt SPM). The 52 and 47 kD proteins were also more intensely phosphorylated in Cd SPM than Cpt SPM. The 100 kilodalton protein, although present in both Cpt and Cd sperm plasma membranes, was found to be phosphorylated at the tyrosine residues only in the Cd SPM, as indicated by the Western blot using antiphosphotyrosine antibody. Further, a differential phosphorylation of the substrate proteins present in the Cpt and Cd SPM was seen when Mg²⁺ in the assay buffer was replaced by other divalent cations. For instance, Zn²⁺ stimulated the phosphorylation of an 85 kD protein in cauda SPM and not in the caput SPM and Ca²⁺ stimulated the phosphorylation of a 76 kD protein only in the cauda SPM. The phosphoproteins in both the plasma membranes were found to be phosphorylated predominantly at the tyrosine residue. The differential phosphorylation of a 100 kD protein at tyrosine in the Cd SPM (Western blot), which is absent in the immature Cpt SPM, also indicated that certain proteins in the hamster spermatozoa are phosphorylated in a maturation-specific manner. Mol. Reprod. Dev. 47:341–350, 1997. © 1997 Wiley-Liss, Inc.     

Direct evidence for the secretion of lactoferrin and its binding to sperm in the porcine epididymis

Lactoferrin has been for the first time purified from the porcine cauda epididymal fluid as a 70 kDa protein. Both Western and Northern blot analyses show that lactoferrin is synthesized in the regions from the distal caput to the cauda epididymis and secreted into the luminal fluid. Lactoferrin is first secreted as a 75 kDa glycoprotein and its carbohydrate moieties are gradually digested to form 70 kDa protein in the cauda epididymis. Lactoferrin has already bound to the surface of the epididymal sperm because the anti-lactoferrin antiserum induces the mature sperm tail-to-tail agglutination. These results strongly suggest new physiological functions of lactoferrin on the sperm maturation in the epididymis. Mol. Reprod. Dev. 47:490–496, 1997. © 1997 Wiley-Liss, Inc.     

Protein D is differentially expressed and regulated in the rat epididymis.

We report the use of a sensitive and specific enyzme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) to study the expression of protein D, a major androgen-regulated sperm-binding glycoprotein at the protein and mRNA level in different anatomical regions of the rat epididymis. The concentration of protein D in the caput, corpus and cauda region of the epididymis was 10.2 +/- 0.67, 7.3 +/- 0.61 and 22.8 +/- 1.34 ng/micrograms total protein, respectively. The total RNA extracted from the caput, corpus and cauda regions of the rat epididymis was amplified by PCR with oligonucleotide primers specific for the 5' and 3' portion of protein D cDNA. Compared to the caput and cauda region, a significant reduction (greater than 82 +/- 3%) in the expression of protein D mRNA levels was observed for corpus epididymal RNA. This data demonstrates regional differences in the concentration of protein D and suggests that protein D expression may be regulated at the level of mRNA within the corpus epididymidis.     

Electron Microscopic (Energy Dispersive X-ray Analysis) Study of Human Male Reproductive Organs and Semen

Recently, technological advancement helped to improve our knowledge on trace elements in human male reproductive organs and its secretion, semen. In this study, employing energy dispersive x-ray analysis facilities on electron microscope, presence of different elements in human male reproductive organs-??testis, epididymis, caput, corpus and cauda, prostate gland, seminal vesicle, Cowper??s gland and vas deferens??seminal plasma and spermatozoa pellet was studied. Several elements were observed. Gold was one among them that was present in seminal plasma and spermatozoa. It was also present in epididymis caput. Authors consider epididymis caput as the source of gold in semen.     

In vivo secretion and association of clusterin (SGP-2) in luminal fluid with spermatozoa in the rat testis and epididymis.

Clusterin (sulfated glycoprotein-2) is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells. An antigenically similar form is synthesized and secreted by the epididymis. The goal of this study was to define the epididymal regions in which clusterin is present and the regions in which clusterin is secreted and interacts with developing spermatozoa. Seminiferous tubule (STF), caput, corpus, and cauda fluids were collected by micropuncture and/or microperfusion and two-dimensional Western blot analysis was performed with a polyclonal antibody directed against Sertoli cell clusterin. Clusterin was found in both STF and epididymal fluid. STF contained predominantly the clusterin heavy chain (45 kd); however, a 70 Kd heterodimer was present under nonreducing conditions. Two subunits of clusterin with lower molecular weights (41 kd, heavy chain; 32 kd, light chain) and higher isoelectric points were present in the luminal fluid of all epididymal regions. The intraluminal levels of the heavy and light chains decreased from caput to cauda. Analysis by two-dimensional gel electrophoresis of proteins secreted directly into the epididymal luminal fluid revealed that clusterin was secreted by caput epithelium and not by the corpus and cauda epithelium. Western blots of membrane extracts from testicular, caput, and cauda spermatozoa revealed that testicular clusterin was associated with testicular sperm and epididymal clusterin with predominantly caput sperm. Our findings suggest that clusterin is secreted into the caput epididymal lumen, where it binds to sperm and then dissociates from sperm to be endocytosed by cells of the distal epididymal epithelium.     

Zona drilling enhances fertilization by mouse caput epididymal sperm

Spermatozoa from the caput epididymis are known to be much less capable of fertilization when compared to sperm from more distal segments of the epididymis. The purpose of this study was to determine if two micromanipulative techniques, zona drilling (ZD) and a modification of partial zona dissection (PZD), could be used to enhance fertilization with caput epididymal sperm. A mouse in vitro fertilization model was used. Inseminating oocytes with 500-1,000 sperm/oocyte from the cauda epididymis as a control resulted in fertilization of 98 of 300 (32.6%) oocytes. Of those fertilized, 47 developed to the blastocyst stage (47.9%). Caput sperm fertilized 13 of 116 (11.2%) nonmanipulated oocytes. Only 1 of 13 developed into a blastocyst, while with oocyte ZD, caput sperm fertilized 24 of 144 (16.7%) oocytes, 50% of those fertilized developing to blastocyst (P = 0.0129). When modified PZD was performed on oocytes, only one of 23 was fertilized, with no blastocyst development. These results indicate that acid Tyrode ZD enhances both fertilization and early embryonal development when caput epididymal sperm are used for insemination. These mouse studies suggest that ZD or other micromanipulation techniques may prove clinically useful in men with proximal epididymal obstruction where only caput sperm are available.