Leona G. Young

Organization of the Human Gene Encoding the Epididymis-Specific EP2 Protein Variants and Its Relationship to Defensin Genes

Abstract The EP2 gene codes for at least nine message variants that are all specifically expressed in the epididymis. These variants putatively encode small secretory proteins that differ in their N- and C-termini, resulting in proteins that can have little or no sequence similarity to each other. We have isolated and sequenced the human EP2 gene to determine the molecular origin of these variants. The EP2 gene has two promoters, eight exons, and seven introns. Exons 3 and 6 encode protein sequences homologous to β-defensins, a family of antimicrobial peptides. This sequence homology and the arrangement of promoters and defensin-encoding exons suggest that the EP2 gene originated from two ancestral β-defensin genes arranged in tandem, each contributing a promoter and two exons encoding a leader sequence and a defensin peptide. The proposed evolutionary relationship between the EP2 gene and defensin genes is supported by the observation that the EP2 gene is located on chromosome 8p23 near the defensin gene cluster and is separated by 100 kilobases or less from DEFB2, the gene for β-defensin-2. While the EP2 gene transcribes β-defensin-like message variants, most of the known message variants code for nondefensin proteins or proteins containing only a partial defensin peptide sequence. We suggest that, during its evolution, the EP2 gene has acquired new functions that may be important for sperm maturation and/or storage in the epididymis.

Duration of spermatogenesis and relative frequency of each stage in the seminiferous epithelial cycle of the chimpanzee

To determine the duration of 1 spermatogenic cycle, a single pulse of tritiated thymidine was infused into a branch of the spermatic artery in each of 3 chimpanzees (Pan troglodytes). Samples were recovered surgically prior to infusion, at 1 h, and at 3, 8, 14, 16, 17, 28, 30, 33, 40, 44, and 48 days postinfusion. Tissues were fixed in Bouins solution, dehydrated, paraffin-embedded, sectioned at 5 micrometers, and stained. Pre-infusion samples were used in morphometric studies to estimate the percentage frequency of area occupied by each of the 6 cellular associations (stages I-VI) characteristic of chimpanzee spermatogenesis, and thus, to estimate the days duration of each stage. To estimate the duration of 1 spermatogenic cycle, pre- and post-infusion, tissue sections were coated with undiluted Kodak NTB2 liquid autoradiographic emulsion and incubated at 4 +/- 1 degree C. At optimum exposure times, slides were processed with Kodak D-19 and Fixer; light microscopic analyses were conducted to determine the most mature labeled cell in stage III for each of the sample times. The duration of the 6 stages (I-VI) are 4.2, 2.0, 4.3, 1.5, 1.3 and 0.6 days, respectively, and the duration of 1 spermatogenic cycle is approximately 14 days. Thus, the duration of spermatogenesis from the Ap spermatogonium to mature Sd2 spermatid is approximately 62.5 +/- 1.5 days or 4.46 spermatogenic cycles.

Immunological identification of lactoferrin as a shared antigen on radioiodinated human sperm surface and in radioiodinated human seminal plasma

After lactoperoxidase-catalyzed iodination of either human sperm surface or human seminal plasma, a 72-76 kdalton component was resolved on SDS-7.5% polyacrylamide gels. The approximate molecular weight of this component was consistent with that of human lactoferrin, a major sperm-coating antigen. Immunoprecipitation of labeled sperm surface and of labeled seminal plasma with specific anti-human lactoferrin antibody identified lactoferrin as a constituent of this 72-76 kdalton component both on human sperm surface and in human seminal plasma. These data show that components which have been inferred to be shared because of similarities in biochemical characteristics also contain immunologically identical components.

Epididymal Specificity and Androgen Regulation of Rat EP2

Abstract In primates, expression of the EP2 gene is androgen-dependent and epididymis-specific. EP2 mRNA expression was investigated in caput, corpus, and cauda regions of rat epididymis and in 15 other rat tissues. Polymerase chain reaction and Northern analyses showed that rat EP2 is expressed predominantly in the proximal caput epididymidis. EP2 mRNA expression was determined in proximal epididymides from castrated, sham-operated, and efferent duct-ligated rats. In castrated rats, EP2 mRNA decreased to <10% of that in sham-operated rats between Days 3 and 4 postcastration, demonstrating the androgen dependence of EP2 expression. In epididymides ligated unilaterally at the efferent ducts, EP2 mRNA levels were approximately equal to those in the unligated contralateral epididymides or in sham-operated rats, indicating that EP2 expression does not depend on testicular factors. In bilaterally castrated rats, immediate and delayed testosterone replacement showed the dependence of EP2 expression on circulating androgens. Injection of testosterone propionate (TP) on Days 0, 1, 2, and 3 postcastration maintained EP2 mRNA levels approximately equal to those in sham-operated rats. Starting at Day 4 postcastration, daily injection of TP for 7 days restored EP2 mRNA to approximately normal levels. These data indicate for the rat that EP2 is expressed specifically in the proximal caput epididymidis and that its expression depends on circulating androgens but not on testicular factors.

Germ cell maturation and cellular associations in the seminiferous epithelial cycle of the chimpanzee

Seminiferous tubule architecture, germ cell maturation steps and cellular associations (stages) of the spermatogenic cycle of the chimpanzee (Pan troglodytes) are resolved. Cross sections of seminiferous tubules usually exhibit 2 to 4 stages, occasionally 1, and rarely 5; stages are not functionally sequential in structurally contiguous regions. The cellular maturation steps are: dark type A stem cell (Ad), pale type A (Ap), type B(B) spermatogonia; resting or preleptotene (P1), leptotene (L), zygotene (Z), pachytene (P), diplotene (Di) primary spermatocytes; meiotic divisions (M1, M2); secondary spermatocytes (2 degrees S); six developmental stages of the spermatid (Sa, Sb1, Sb2, Sc, Sd1, Sd2) composing spermiogenesis. The germ cell maturation steps characteristic of the six cellular associations (stages I-VI) are: Ad, Ap, B, P, Sa, Sd1 (I); Ad, Ap, B, PI, P, Sa, Sd2 (II); Ad, Ap, B, PI, L, P, Sb1 (III); Ad, Ap, PI, L, P, Sb2 (IV); Ad, Ap, L, Z, P, Di, Sc (V); Ad, Ap, B, Z, P, Di, 2 degrees S, Sc (VI). Surgical pressure trauma causes sloughing of some 2 degrees S spermatocytes and some Sa, Sb1, Sb2, Sd1, and Sd2 spermatids, resulting in missing generations, and disrupts Sertoli cell attachments, affecting germ cell development and associations. In structure and function, chimpanzee spermatogenesis appears most similar to the human.

Viscometric analysis of the contractile proteins of mammalian spermatozoa.

Abstract Modifications of the methods for isolating myosin, actin, and actomyosin from mammalian muscle were employed to extract the contractile proteins of the bull spermatozoan flagellum in order to study their physicochemical properties. The flagellum was found to contain at least two distinct proteins, flactin and spermosin, which interacted to form a “complex”, flactospermosin, deformable in the presence of ATP and divalent cations. Bull sperm flagellar spermosin resembled muscle myosin in that it showed no viscometric response to the addition of ATP and magnesium ions, but did interact reversibly with both actin and flactin. Bull sperm flagellar flactin resembled muscle actin in that it contained a stoichiometric quantity of bound nucleotide, was polymerizable in the presence of ATP and MgCl 2 , was depolymerized in the presence of KI, and interacted reversibly with both myosin and spermosin. Flactospermosin, both when extracted directly from the spermatozoa or when prepared by mixing flactin with spermosin, responded to the presence of ATP and magnesium ions by undergoing a reversible decrease in viscosity.

Sequential histology of the adult chimpanzee epididymis

The chimpanzee epididymis consists of three gross anatomical regions: caput (Cp), corpus (Cr), and cauda (Cd). During epididymal transit, chimpanzee sperm undergo maturational changes (affecting motility, capacitation, and the acrosome reaction), which enable fertilization. Elucidation of the sequential histology of the chimpanzee epididymis is preliminary to determining the role of each region of epididymal duct in the process of sperm maturation. We report the histology, in the adult male chimpanzee, of sequential segments of epididymis from the proximal efferent ductules through the Cp, Cr, and Cd regions of the epididymis proper to the proximal ductus deferens. These data illustrate the gross architecture of the tissue and reveal the histology characteristic of the diverse epithelia composing the epididymal ducts at each level. The tubules composing the epididymis of the adult male chimpanzee present at least 16 histologically-distinct epithelia and their transitional forms. Such diversity of epithelia suggests a corresponding diversity of function. Although there are some clear differences, the histological appearance of the ducts, and of the component epithelia of the chimpanzee epididymis, is remarkably similar to that reported for the human.

EP2 Splicing Variants in Rhesus Monkey (Macaca mulatta) Epididymis

Abstract The expression pattern of EP2 variants was examined in the rhesus monkey (Macaca mulatta). Using reverse transcriptase-polymerase chain reaction and rapid amplification of complementary cDNA protocols, 11 message variants were identified in rhesus epididymis, only three of which (EP2B, EP2C, and EP2E) have previously been reported. The most abundant variant found in human, EP2A, was not found in rhesus. Seven of the eight new rhesus EP2 variants (EP2J–EP2Q) use previously unidentified 5′-splicing sites in exon 3, and four variants use three previously unidentified exons whose counterparts are present in the human EP2 gene. Overall, 3 of the 11 variants, EP2C, EP2E, and EP2Q, code for β-defensin-like peptides whose probable physiological role is to protect the male reproductive tract against microbial invasions. Because of the complex splicing pattern that causes some downstream exons to be read in any of the three reading frames, the N-termini of the other eight EP2 peptide variants consist of a partial β-defensin motif with three cysteines, followed by amino acid sequences that have no recognizable homology to known proteins.

Functional parameters of chimpanzee (Pan troglodytes) sperm from ejaculates collected by rectal probe electrostimulation and by artificial vagina

This study compares functional parameters of sperm from ejaculates collected from 15 adult male chimpanzees using rectal probe electrostimulation (RPE) and from 10 adult male chimpanzees trained to use an artificial vagina (AV). Computer assisted motion analysis (CAMA) showed no significant differences in mean values for straight line velocity (VSL), linearity (LIN), curvilinear velocity (VCL), and lateral head movement (ALH) of sperm from ejaculates collected by RPE and by AV. There was, however, a significant difference (P < 0.01) in the population distribution for VSL and LIN, which indicates that sperm swim in a more convoluted manner in ejaculates collected by RPE than in ejaculates collected by AV. In the hamster zona‐free ovum penetration assay (SPA), there were no significant differences in the percentages of hamster oocytes penetrated by sperm or in the number of sperm which penetrated each oocyte after 4 or 24 h incubation using sperm from ejaculates collected by RPE and by AV. Therefore, the lack of success using sperm from ejaculates collected by RPE to initiate pregnancy in the chimpanzee does not appear to result from abnormalities in sperm fertilizing capacity as measured in SPA.

Estimate of epididymal transit time in the chimpanzee.

During their passage through the epididymis, sperm undergo functional changes which result in their maturation and in their ability to fertilize ova. Maturational changes effected during epididymal transport are attributable to sequential changes in various regions of the plasmalemma of the sperm head and flagella. These functional changes in the plasmalemma result, at least in part, from the sequential binding of proteins secreted by the epididymal epithelium into the epididymal lumen. An estimate of epididymal transit time is essential to such investigations. Time elapsed from a testicular arterial infusion of a single pulse of tritiated-thymidine to the release of 3H-labeled sperm into the lumen of the seminiferous tubule was about 39 days. Seminal fluid-free 3H-labeled sperm first appear in the ejaculate about 41 +/- 1 days post-infusion. Total transit time for 3H-labeled Sd2 sperm released into the tubular lumen to appear in the ejaculate is estimated as the difference between these values. Since total transit time is equal to the seminiferous tubule transit time plus the epididymal transit time, epididymal transit time constitutes some lesser portion of the total transit time of 2 +/- 1 days.