Dennis Lacy

ANDROGEN DEPENDENCY OF SPERMATOGENESIS AND THE PHYSIOLOGICAL SIGNIFICANCE OF STEROID METABOLISM IN VITRO BY THE SEMINIFEROUS TUBULES

Summary It has long been known that spermatogenesis is responsive to various steroids under certain experimental conditions. The evidence for this is briefly reviewed alongside more recent data which have shown that the seminiferous tubules of the normal rat contain proteins, with characteristics similar to those found in the prostate, which specifically bind testosterone. It is concluded that spermatogenesis is normally dependent on androgens without which there is a marked failure at meiosis. Evidence that the seminiferous tubules and interstitial tissue of rat testes can be separated by dissection without significant cross contamination is considered. Some recent studies are described in which, by the use of light and electron microscopy and a differential 17β-hydroxysteroid dehydrogenase response, it has been demonstrated that the technique can be applied successfully to human tissue. The physiological significance of steroid metabolism by the tubules in vitro is then examined with particular reference to (a) relationship between cholesterol metabolism and spermatogenesis under normal and experimental conditions and the ability of the tubules of the rat to effect side chain cleavage of the cholesterol molecule in vitro (b) relative ability of the tubules of the rat to metabolise progesterone to testosterone in vitro as compared with that shown by the interstitium with the microsomal fraction of the former displaying equal or even higher activity than the latter per unit wet weight of tissue (c) correlation between events occurring in vivo and changes in steroid metabolism by the tubules in vitro with particular reference to spermatogenesis in a seasonal mammal (d) biochemical and morphological studies on tubules in which the ratio of Sertoli cells to germ cells was altered experimentally or underwent a natural change (e) studies on steroid metabolism in vitro by the tubules of the human testes and (f) effects of FSH and LH on endogenous levels of testosterone and steroid metabolism in vitro by the tubules and interstitium of the rat testis. It is concluded that the ability of the tubules to metabolise steroids in vitro is of physiological significance and that they are a source of androgens, probably produced by the Sertoli cells under the influence of FSH, for promoting and maintaining spermatogenesis. Attention is focussed on the possibility that metabolites found in the tubules may influence other parts of the reproductive tract to which they have ready access via the duct system.

Transmission electron microscopy and the production of steroids by the Leydig and Sertoli cells of the human testis

Abstract An account is given of the fine structure of human Leydig and Sertoli cells using tissue in which, from parallel kinetic incubation studies to be reported upon elsewhere, it was known that the interstitial tissue and seminiferous tubules each possessed the capacity to elaborate androgen in vitro . In confirmation of previous work, the mature Leydig cells were observed to contain abundant amounts of smooth-surfaced endoplasmic reticulum in the form of discrete vesicles. The mitochondria often showed small cup-like invaginations of their outer surfaces into which occasionally extended vesicles of the smooth-surfaced endoplasmic reticulum. The lipid, which, in corresponding light microscope preparations gave a positive reaction to the Schultz test for unsaturated sterols, was almost always associated with a dense granular material; there was no morphological evidence to suggest that these composite inclusions were involved in steroid synthesis. The cells contained occasional bundles of fine filaments and crystals of Reinke. The amount of smooth-surfaced endoplasmic reticulum varied in different regions of the Sertoli cells but was most abundant in areas containing many lipid droplets (Schultz-negative), numerous mitochondria and parallel arrays or stacks of granular endoplasmic reticulum. In these cells the smooth-surfaced endoplasmic reticulum was mainly in the form of interconnected tubules. The mitochondria were often conspicuously grouped around the lipid droplets which appeared to be in various stages of assimilation. The stacks of granular endoplasmic reticulum were orientated towards the lipid droplets and opened into tubules and cisternae of the smooth-surfaced endoplasmic reticulum. When considered alongside cell-fractionation studies on the human testis, and the results of the parallel incubation studies referred to above, it is concluded that the present work provides additional evidence that both the Leydig cells and Sertoli cells are steroidogenic. The simultaneous demonstration of both the vesicular form (Leydig cells) and tubular pattern (Sertoli cells) of the smooth-surfaced endoplasmic reticulum, suggests that both appearances were genuine and may reflect upon different physiological states of the cells. As far as could be judged morphologically, the Sertoli cells appeared more active than the Leydig cells.

Application of scanning electron microscopy to semen analysis of the sub-fertile man utilising data obtained by transmission electron microscopy as an aid to interpretation

Abstract The limitations of semen analysis as routinely practiced by light microscopy are briefly reviewed. The value of using SEM together with information obtained from TEM has been examined in a ‘blind’ study on three men attending a sub-fertility clinic so that clinical details, including sperm concentration, were not known to the investigators until the work was completed; the men had a clinical history of infertility and the sperm concentrations of the samples examined by electron microscopy were 16 × 10 6 /ml, 84 × 10 6 /ml and 237 × 10 6 /ml. For study by SEM the cells were fixed in buffered glutaraldehyde, concentrated by low-speed centrifugation and tried with acetone, collected on a Millipore filter, dried by the critical point method and coated with gold. This method of preparation gave excellent preservation. Micrographs were obtained showing up to 40 spermatozoa per field which were suitable for assessing variations in the general shape and size of the cells and, at high magnification, differences in surface texture. All the samples contained spermatozoa possessing a large mass of extraneous cytoplasm which was readily visible by SEM. When examined by TEM this region possessed features characteristic of the residual cytoplasm (or body) of spermatids. This results, together with others reported in the present paper and data published elsewhere, suggests that sperm dysgenesis may be a relatively common feature of the spermatozoa of sub-fertile men. It is suggested that the degree of differentiation could be used as an objective basis for identifying and hence classifying, cells considered to be abnormal. It is concluded that SEM, together with information obtained by TEM, can make a valuable contribution to semen analysis of sub-fertile men with widely different sperm concentrations.

Biological applications of combined transmission electron microscopy and X-ray microanalysis with special reference to studies on the mammalian testis

Abstract The main advantages of being able to combine high resolution electron microscopy with X-ray probe microanalysis, as at present exemplified by EMMA 4, are essentially as follows: 1. chemical information can be obtained in situ ; 2. the detector system is highly sensitive (10 −17 g) and accurate, providing both qualitative and quantitative data; 3. ease of operation, comparable to the use of a conventional instrument. However, in selecting the type of problems which can be investigated, it is essential for biologists to recognise the following limitations or difficulties: 1. elements are detected as distinct from compounds; 2. at the present stage of development, weak X-ray signals from elements with an atomic number below 11 cannot be detected; this rules out H(1), C(6), O(8), and N(7); 3. detection is governed by the amount of the element in the volume being analysed; with ultra-thin sections this volume is only about 0.003μm 3 ; 4. changes may be induced in the specimen during the period of examination; 5. difficulties associated with the use of freezing techniques to simulate in vivo conditions. Depending upon the nature of the problem some of these limitations can be avoided or minimised. Three main types of general application are discussed: 1. functional aspects based on elemental analysis; 2. studies on the relative concentration of particular elements and 3. as an alternative to established ‘tracer’ techniques. To illustrate the merits of EMMA 4 when applied to particular problems (e.g. the study of a mixed population of cells) the results of some preliminary observations on the testes of a normal man and of normal and experimentally treated rats are reported. It is concluded that one outstanding feature of ‘fine structure analysis’ is the ability to demonstrate a chemical change in the absence of any morphological criteria; this has great significance in both experimental work (e.g. in studying the effects of hormones) and in diagnosis, particularly when only small biopsy specimens may be available.

NORMAL STRUCTURE OF SPERMATID NUCLEI AND CHANGES CAUSED BY IONIZING RADIATION

SummarySpermatid nuclei, at a certain period of their development, have been examined both in normal cells and in those which have been irradiated with a dose of 10 000 rad using 14 mev electrons. Within normal nuclei we have identified two main components: a nuclear matrix and chromosomes. The chromosomes consist essentially of a chromosomal matrix of low electron density and microfibrils. The latter are about 25 A thick and are coiled. The gyre-width and pitch of such coils is about 100 A and 75 A respectively. Throughout much of the chromosomes the microfibrils are apparently arranged in the form of an open network in which some semblance of a hexagonal pattern can sometimes be discerned. In some regions the microfibrils are extremely numerous and closely packed together to form multi-stranded units. Such regions stain positively by the Feulgen technique. Coiled microfibrils also occur in the nuclear matrix. Three days after irradiation some nuclei superficially appear to be only slightly damaged. Howe...

Isolation of residual bodies and spermatid cytoplasm from the testis of the adult rat

Abstract In an attempt to obtain improved separation of residual bodies of rat testes consistent with good preservation of morphological detail, the tissue was chopped to avoid a shearing action, suspended in 0.25M sucrose buffered at pH 7.4 and filtered. The filtrate was centrifuged, the pellet suspended in buffered 0.25M sucrose and layered on the upper surface of a discontinuous sucrose gradient consisting of 0.8M, 1.0M and 1.9M sucrose. Three fractions were collected: Fraction 1 at the 0.25M/0.8M interface, Fraction 2 from the 0.8M region andFraction 3 at the 0.8M/1.0M interface. Samples of each fraction were examined by electron microscopy; Fractions 1 and 2 consisted mainly of residual cytoplasm at a late stage ofdevelopment or residual bodies, while Fraction 3 consisted mainly of spermatid cytoplasm at an earlier stage.