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Treatment of rabbit spermatozoa with Triton X-100 and dithiothreitol for increasing periods of time results in progressive increases in both the percentages of spermatozoa with swollen nuclei and in the ability of the swollen nuclei to serve as template for DNA synthesis. The involvement of endogenous proteolytic activity in this thiol-induced swelling and release of template restrictions is suggested by the fact that when spermatozoa are incubated in solutions containing various inhibitors of proteolysis (soybean trypsin inhibitor, lima bean trypsin inhibitor, or p-aminobenzamidine) in addition to Triton X-100 and dithiothreitol, inhibition of nuclear swelling and DNA template activity occur. The degree of inhibition is dependent upon the concentration of inhibitor employed. These results demonstrate a direct relationship between release of DNA template restrictions and nuclear swelling, and suggest that both processes depend upon some degree of endogenous proteolytic activity as well as upon disulfide reduction.

Autoradiographic and electron microscope methods were used to correlate changes in nucleoproteins with nuclear fine structure during spermatogenesis in the mouse. Testes were fixed at daily intervals after intratesticular injectionwith labeled amino acid. [3H]Arginine, lysine, valine, and proline were rapidly incorporated into primary spermatocyte nuclei, retained through subsequent spermatocyte divisions and through spermatid differentiation to step 12 of spermiogenesis, but were lost with spermatid differentiation beyond step 12. Arginine and lysine (not valine or proline) also were rapidly incorporated into certain elongated spermatid nuclei but differed strikingly in their distribution and fate. Nuclei of late step-12 through step-15 spermatids were initially labeled with arginine. This label was retained through subsequent spermatid differentiation and sperm maturation in the epididymis. By contrast, lysine was initially incorporated only into late step-12 and step-13 spermatid nuclei, and was retained only to early step 14 of spermiogenesis. Spermatid incorporation of lysine coincided with the initiation of chromatin condensation in late step-12 nuclei, and loss of lysine coincided with the completion of condensation in step-14 nuclei.

An ovarian in vitro perfusion method was adapted to examine rabbit corpus luteum (CL) function during pseudopregnancy. Ovaries were perfused in vitro with tissue culture Medium 199 with or without 3% bovine serum albumin (BSA). Samples were obtained from both arterial and venous cannulae, allowing rates of progesterone secretion to be determined. Two perfusion methods were compared: a closed system in which perfusion medium continuously recirculated through the tissue, and an open system in which the ovarian vein cannula was left outside the perfusion chamber. The addition of 3% BSA was found to prevent edema and distortion of the interstitial space, and to result in increased progesterone secretion. With the closed perfusion system, the progesterone secretion rate measured over the 6-h perfusion was significantly higher on Day 11 than on Day 1 of pseudopregnancy and had declined significantly on Day 18. There was no difference in secretion rates whether the perfusion system was open or closed, and the results obtained with both systems closely approximated in vivo progesterone secretion rates. Measurements of CL tissue progesterone content before and after in vitro perfusion indicated that the changes in progesterone secretion seen during pseudopregnancy resulted from differences in the synthesis and secretion of progesterone and not from leakage of progesterone already present in the CL prior to perfusion. Taken together, these results indicate that the modified in vitro perfused rabbit ovary preparation described herein is an appropriate model to examine progesterone secretion by ovaries bearing CL.

We employed stereological methods at the light-microscope level to examine the mechanism by which corpora lutea (CL) grow during the course of pseudopregnancy in the rabbit. Corpus luteum volume per ovary, the absolute volume of luteal cells per CL, individual luteal cell volume, the number of luteal and endothelial cells per CL, and capillary surface area per CL were examined in rabbits at Days 1, 4, 7, 11, and 18 of pseudopregnancy. Total CL volume increased from 3.7 ± 0.1 μl to 30.3 ± 0.5 μl over Days 1 to 11 and thereafter decreased to 15.2 ± 1.1 μl by Day 18. Stereological analyses showed that the increases in CL volume from Day 1 to Day 11 were due primarily to increases in the volume of individual luteal cells (from 2.6 ± 0.2 pl on Day 1 to 23.5 ± 1.7 pl on Day 11, 1 pl = (10 μ)3; r = 0.96), and that the decrease in CL volume after Day 11 resulted largely from a decrease in luteal cell volume (to 12.8 ± 1.5 pl). In contrast, no change was seen in the number of luteal cells per CL (range 9.1 × 105−12.5 × 105). These data show that CL growth and subsequent regression during pseudopregnancy result primarily from changes in the volume of individual luteal cells, and not from changes in the number of luteal cells. These data support the hypothesis that modulation of progesterone production during pseudopregnancy is due to changes in individual luteal cell volume and not to changes in cell number.

The present studies were designed to identify mouse spermatid proteins into which intratesticularly injected [3H] arginine and [3H] lysine were initially incorporated and to determine the fates of those proteins during subsequent spermatid differentiation. At intervals between 2 h and 7 days after injections, elongated spermatid nuclei were isolated from the testes by virtue of their resistance to sonication, and mature sperm nuclei were isolated from the epididymides. Basic proteins were extracted from isolated spermatid and sperm nuclei and subjected to electrophoresis on acid-urea polyacrylamide gels. Two hours after injection, [3H] arginine was seen in a number of spermatid basic proteins, including both the “testis-specific” protein (TP) and the protamines. As expected from previous studies, only one class of these labeled proteins, protamine, was retained through the completion of spermiogenesis and sperm maturation 7 days later. In striking contrast, [3H] lysine was initially incorporated only into the spermatid TP protein, was retained for only 3 days, and was then lost. Our previous autoradiographic study (Mayer and Zirkin, 1979) demonstrated that intratesticularly injected [3H] lysine was initially incorporated into elongating spermatid nuclei at the initiation of chromatin condensation (late step 12 and step 13), was retained for 3 days through the completion of chromatin condensation (step 14), and was then lost. The present results, taken together with the results of our previous autoradiographic study, demonstrate striking temporal relationships between the first appearance of newly synthesized TP protein and the initiation of chromatin condensation in spermatid nuclei of late step 12, and between the loss of TP protein and the completion of chromatin condensation in spermatid nuclei of step 14.

Rat and hamster testes, epididymides, vas deferentia, seminal vesicles, prostates and coagulating glands were assayed for sulfhydryl oxidase activity. The activity, present primarily in the fluids of these organs, was lowest in the testis, increased through the epididymis and was highest in the seminal vesicle. The localization and reactivity of sulfhydryl oxidase activity suggests that it may play a role in regulating the degree of disulfide bonding in spermatozoa during epididymal maturation and storage and after ejaculation.