Eleanor B. Smithwick

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.

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.

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.

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.

Extraction of ATP-phosphohydrolase activity from epididymal bull sperm and ultrastructure of flagellar degradation☆

Abstract ATP-phosphohydrolase activity was solubilized from isolated epididymal bull spermatozoa flagella by a variety of extractants. The effects of isolation and chemical degradation on specific structural components within the flagellum were visualized electron microscopically. Low ionic strength solutions solubilized 30% of the ATPase activity and 10% of the protein content. High ionic strength KCl solubilized 50% of the ATPase activity and 10% of the protein content. Distinctions between mechanical effects of isolation and mechanical and chemical effects of extraction were determined. As demonstrated by transmission electron microscopy while 10 mM Tris/250 mM sucrose, pH 7.4, caused slight and generalized extraction of all flagellar components, alkaline 0.5 M KCl caused increased discontinuity and disorganization of flagellar structure as well as the solubilization of specific flagellar structures, including B and central pair microtubules, dynein, nexin, radial arms, secondary fibers and an extractable matrix from the outer dense fiber/satellite fiber complex. The A microtubules of the axoneme and the substructure of the outer dense fiber/satellite fiber complex exhibited similar structural stability. Simple criteria for reducing subjectivity in ultrastructural data are proposed.

Effects of mechanical and chemical disruption on the ATP-phosphohydrolase activity and ultrastructure of sperm flagella☆

Abstract The Ca2+- and Mg2+-ATP-phosphohydrolase (ATPase) activities measurable in suspensions of and extracts from bull epididymal spermatozoa flagella, sonicated for 2 min at 8 W, were compared with those of control flagella after 0, 3, and 18 h dialysis against an alkaline 0.5 M KCl extracting solution. Activity was measured in the absence of and in the presence of oligomycin. The effects of sonication and extraction on structural components within the flagella were visualized electron microscopically. Sonication caused fragmentation of flagella, extensive disruption of mitochondria and an immediate (0 h) increase in both Ca2+- and Mg2+-ATP-ase activity. Prolonged dialysis resulted in solubilization of specific flagellar structures, partial disruption of mitochondrial integrity, and increases in ATPase activity. Mg2+-ATPases of flagellar suspensions and extracts were greater than Ca2+-ATPases, and a part of this Mg2+-ATPase activity was inhibited by oligomycin. Therefore, Mg2+-ATPases from disrupted mitochondria contribute to the Mg2+-ATPase activities measurable in suspensions of and extracts from bull sperm flagella. This study emphasizes the necessity of evaluating the effects of the mechanical and chemical treatments used in fractionating cells before interpreting the biochemical information derived from their isolated components.