Spermatozoa are produced in the testis and undergo post-gonadal modifications in the epididymis to acquire fertilizing ability. In epididymal plasma, high-molecular-weight proteins and such small molecules as free-L carnitine convert the gametes into "competent' and functional cells. This review summarizes the knowledge pertaining to L-carnitine and the significance of free L-carnitine uptake into the mature spermatozoa of mammals. We provide an overview of the function of free L-carnitine and carnitine esters in the metabolism of eukaryotic cells and review the role of the specific carnitine acyltransferases in mitochondrial transport of fatty acids and in modulating acyl-coenzyme A (CoA) pools in cellular organelles. In mammals, including man, free L-carnitine is taken from blood plasma and concentrated in the epididymal lumen. This epididymal secretion is beneficial for spermatozoa and is not merely an excretory waste. The uptake of free L-carnitine into the spermatozoa and its metabolic outcome are discussed first in in-vivo and then in in-vitro situations. Free L-carnitine goes through the sperm plasma membrane by passive diffusion. Free L-carnitine is acetylated in mature spermatozoa only. The excess acetyl-CoA from the mitochondria is probably stored as acetyl-L-carnitine and modulates the reserves of free CoA essential to the function of the tricarboxylic acid cycle. These properties of L-carnitine of buffering CoA in the mitochondrial matrix are known in somatic cells but are accentuated in this study of the male germinal cells. In the future, a precise measurement of the in-vivo and in-vitro concentrations of free CoA and acetyl-CoA in the cellular compartments of immature and mature spermatozoa might complete these data. The relationship between the endogenous pools of free and acetylated L-carnitine and the percentage of progressive sperm motility indicates a more important metabolic function related to flagellar movement. In conclusion, the potential to initiate sperm motility, which takes place in the epididymis, is probably independent of the carnitine system, while the energy properties of acetyl-L-carnitine can only be relevant in situations of "energy crisis'. The uptake of "cytoplasmic' free L-carnitine in mature spermatozoa must be a protective form of mitochondrial metabolism, useful to the survival of this isolated cell.

Radioactive myo-inositol was injected intraperitoneally into nephrectomized rats. The radioactive material present in liver, spleen, brain, heart, diaphragm, seminal vesicle, coagulating gland, prostate, epididymis, vas deferens and testis was shown to consist exclusively of myo-inositol and its derivatives, as shown by paper chromatography of hydrolysates and trichloroacetic acid extracts of these tissues. Radioactive myo-inositol was accumulated rapidly within 1 h by the thyroid, coagulating gland and seminal vesicle. Other tissues, such as the pituitary, prostate gland, liver and spleen, concentrated myo-inositol less actively. The muscle tissues studied (diaphragm and heart) concentrated little inositol, whereas brain, testis, and epididymal fat-pad did not concentrate it at all. The lipid fraction of liver contained most of the radio-labelled myo-inositol. In the other organs most of the radioactivity was found in the aqueous trichloroacetic acid extract, largely as free myo-inositol.

Inasmuch as caput epididymal and even testicular spermatozoa are now being used to generate pregnancies by direct injection into the oocyte, differences in the chromatin of spermatozoa from proximal and distal locations in the epididymis were studied. Acridine Orange staining was used to investigate chromatin structure in human spermatozoa which had left the testis and were undergoing maturation in the epididymis. Measurement of green and red fluorescence intensities of human spermatozoa by flow cytometry demonstrated a decrease in binding of Acridine Orange to DNA as the spermatozoa traversed the epididymis. Using spermatozoa from the cauda epididymis as the standard, the percentages of spermatozoa from the efferent duct, proximal corpus epididymis, midcorpus epididymis, distal corpus epididymis, proximal cauda epididymis and distal cauda epididymis that had matured with regard to chromatin condensation were 28 +/- 5, 39 +/- 3, 49 +/- 5, 64 +/- 5, 69 +/- 6 and 74 +/- 4% respectively. It may be concluded that eggs fertilized by ejaculated spermatozoa receive a more highly condensed form of chromatin than that received by eggs inseminated with proximal epididymal or testicular spermatozoa.

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The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality.

The method described may serve as a simple, non-invasive and reliable assay to help clinicians counsel patients with severe male infertility before referring them for testicular surgery to locate spermatozoa for ICSI.

SUMMARY Changes in the lipids of soybeans brought about by Rhizopus orysae during the production of tempeh were studied. The mold possesses strong lipase activity and caused the hydrolysis of over one‐third of the neutral fat of the soybean during the three‐day fermentation. The fatty acid composition of soybean tempeh was compared with that of cooked soybeans by vapor‐phase chromatography of the methylesters. The neutral fat was composed of palmitic, stearic, oleic, linoleic, and linolenic acids, with linoleic acid predominating. These acids were liberated during fermentation in roughly the same proportions found in soybeans after heating 90 min at 100°C. During the most active mold growth, proportionately higher levels of palmitic acid were found, and the level of linoleic acid was somewhat lower. Except for the depletion of some 40% of the linolenic acid in the later stages of the fermentation, there apparently was no preferential utilization of any fatty acid.

Human sperm were found to contain acetylcarnitine, carnitine, and only traces of propionylcarnitine and four-carbon acylcarnitines. Carnitine and acetylcarnitine were present in higher concentrations in sperm than in the corresponding seminal fluid samples, and the degree of acylation of carnitine was greater in sperm than in seminal plasma. The ratio of acetylcarnitine/carnitine was 1.77 +/- 0.69 in extracts of sperm from samples with a low degree of motility (0-10% motile), whereas it was 4.70 +/- 1.58 in samples which were 40-80% motile. The possible significance of this difference with regard to the degree of acylation of coenzyme A is discussed.