The triphasic nature of Leydig cell development in humans, and comments on nomenclature.

Leydig cell development in humans, although for years described as being biphasic, with fetal and adult phases of maturation, is better considered as a triphasic developmental phenomenon. The morphological literature is summarized in this commentary. Although the majority of studies are of a qualitative nature and many questions remain as to the relative and absolute numbers of cells involved in these developmental phases, this literature is more consistent with a triphasic developmental pattern. This view of Leydig cell development is in accord with the well-known triphasic history of testosterone production, i.e. peaks at 14-18 weeks of fetal life, 2-3 months after birth, and from puberty throughout adult life. It is also significant that the neonatal phase of testosterone production is dependent upon reactivation of the hypothalamic-pituitary-testicular axis (HPT). The current interest in the functional implications of the neonatal period will be better served by considering human Leydig cell development as triphasic.

Mitochondrial cristae diversity in human Leydig cells: a revised look at cristae morphology in these steroid-producing cells.

Mitochondria of steroid-producing cells are integrally involved with steroidogenesis. For decades, the mitochondrial morphology of Leydig cells, as with other steroid-producing cells, has been known to differ from typical mitochondria in that the cristae are predominately "tubular." In a few species, humans being one example, the cristae have often been further categorized as "tubular and/or lamellar," without further elaboration. In the present study, mitochondria of human Leydig cells were examined with the purpose of providing a more detailed description of "cristae" morphology in these steroid-producing cells. The cristae are found to be rather diverse in morphology, consisting of elements of anastomosing tubules in continuity with small cisternal regions as well as with stacked arrays of lamellae, referred to as "lamellar associations." The tubular elements are found to branch in a tripartite fashion and sometimes to expand into small cisternal elements at these junctures. The lamellar associations are a distinctive feature of cristae in human Leydig cells and consist of two to eight closely apposed lamellae with a consistent gap of approximately 4 nm between the membranes of apposing lamellae. Such a close association of cellular membranes is highly suggestive of an integral transmembrane linkage. Although the lamellar associations often appear isolated, evidence is present of a continuity of this compartment of the cristae with the tubular elements. The connections (termed "initial segments") of the various forms of the cristae to the inner mitochondrial membrane are typically via tubules. Mitochondria exhibiting a central region of matrix delineated by one or more cup-shaped lamellae are also present. The pleomorphic structure of mitochondrial cristae in human Leydig cells reemphasizes our present lack of knowledge of how subcellular structure relates to steroidogenesis.

Blockade of the hypothalamic-pituitary-testicular axis with a GnRH antagonist in the neonatal marmoset monkey: changes in Leydig cell ultrastructure.

Little is known of the cell biology of Leydig cells during the neonatal activation of the hypothalamic-pituitary-testicular (HPT) axis. The current study examined the effect of blockade of the HPT axis with a GnRH antagonist (antide) on the neonatal population of Leydig cells in the new world primate, the common marmoset. Three sets of twins, age 7 weeks, were studied: in each pair one twin was used as a control, while the other received treatment with GnRH antagonist from the day of birth to suppress pituitary gonadotrophin secretion. Leydig cells of treated animals were dramatically different from those of controls. The cells were atrophic and exhibited very irregular nuclei. The organelles involved in steroid synthesis were reduced to the extent to being barely evident. The smooth endoplasmic reticulum (SER) was greatly diminished in quantity and distribution. The usual form of the SER (anastomosing tubules) was not evident, but, instead, the SER was relatively unbranched. Peroxisomes, organelles involved in transfer of cholesterol to the mitochondria, were greatly reduced in number. Mitochondria were relatively sparse and exhibited a non-typical morphology, as tubular elements of the cristae were rarely evident. Thus, the central apparatus in steroid production, the SER, mitochondria and peroxisomes, was essentially shut down in the GnRH-antagonist-treated animals. Storage of cholesterol, the precursor of steroid biosynthesis, was also not in evidence, as lipid droplets were extremely rare. Two prominent features of control in neonatal marmoset Leydig cells, the membranofibrillar inclusion (MFI) and basal laminae, remain prominent in the Leydig cells of treated animals. Evidence of apoptosis was not observed. These results provide strong support that the gonadotrophic hormones are the primary regulator of neonatal Leydig cell development in primates, and also suggest cell regression, rather than apoptosis, being the mechanism of this inhibition.

Ultrastructural evidence of adrenergic, as well as cholinergic, nerve varicosities in relation to the lamina propria of the human seminiferous tubules during childhood.

The ultrastructure of autonomic nerve fibers and terminal varicosities is described in relation to the lamina propria of the human seminiferous tubules during childhood (age 3 to 10 years). Autonomic nerve varicosities are classified as: Type I with numerous small (30-60 nm) agranular vesicles and variable numbers of large (100 nm) granular vesicles, and Type II with numerous small (30-60 nm) granular vesicles and sporadic large granular vesicles. These two varicosity types are consistent in morphology with cholinergic and adrenergic nerve terminals, respectively. Nerve varicosities are found, associated with Schwann cells, in proximity to the cells of the lamina propria. Although not found in direct "synaptic' contact, these autonomic endings are often within a few hundred nanometers of the cellularity of the lamina propria. The Schwannian sheath is interrupted over the varicosities at these sites and occasionally the terminal varicosities are totally lacking a Schwann sheath. These findings are consistent with the structural relationship of autonomic nerve "terminals' and effector in other endocrine and non-endocrine systems. This is the first evidence of adrenergic nerve varicosities in proximity to the lamina propria in humans (at any age). Evidence is also presented which suggests a locational difference in the distribution of cholinergic (Type I) and adrenergic (Type II) nerve varicosities in this region, with only cholinergic endings observed directly adjacent to the basal lamina of the seminiferous tubules.

Ultrastructural evidence of indirect and direct autonomic innervation of human Leydig cells: comparison of neonatal, childhood and pubertal ages.

Recent physiological studies have indicated an autonomic influence on the secretion of testosterone from Leydig cells in humans and laboratory animals. Furthermore, a few studies have shown enhanced autonomic control of Leydig cell function in immature, relative to mature, laboratory animals. In the current ultrastructural study of the human testicular interstitium the morphology of autonomic components is described from neonatal, childhood and pubertal ages. Autonomic nerve fibers and varicosities with neurotransmitter vesicles are described in proximity to Leydig cells. The observed autonomic terminals are classified by vesicle morphology into three general types: (1) Type I with predominantly small agranular vesicles (30-60 nm) and occasional larger granular vesicles (100 nm). This type is morphologically consistent with being cholinergic. (2) Type II with predominantly small granular vesicles (30-60 nm), as well as sporadic large granular vesicles. These are morphologically consistent with adrenergic terminals. (3) Type III which exhibit numerous large granular vesicles of mixed size. Evidence of autonomic terminals is encountered most frequently in childhood biopsies, age 3 to 10 years. The neonatal specimen (4 months) is noteworthy in that many of the Schwann cells appear immature and no adrenergic terminals are observed. In contrast, terminals morphologically consistent with being adrenergic are common in the childhood series of biopsies. Although the vast majority of the autonomic terminals are associated with Leydig cells indirectly as "boutons en passant", separated by approximately 150 nm to more than a micron, evidence of direct contact (20 nm) of autonomic terminals with Leydig cells is presented.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural evidence of mature Leydig cells and Leydig cell regression in the neonatal human testis.

The neonatal period in male development is characterized by an acute rise in serum testosterone, which peaks at 2 to 3 months of age. The purpose of this study is to examine the neonatal human testicular interstitium at 4 months for evidence of Leydig cell maturation, as well as any morphological criteria relating to the fate of Leydig cells during this period, specifically, for signs of cell regression. Leydig cells are described with impressive development of the steroid secreting apparatus, which are consistent with the mature Leydig cells found during early fetal development and in the adult. The outstanding feature of these cells is the "organelle association" of extensive, anastamosing tubules of smooth endoplasmic reticulum (SER), pleomorphic mitochondria with a component of tubular cristae, and abundant microperoxisomes associated with the SER. Well-developed Golgi elements, regionalized RER, and diverse cell inclusions are also characteristics of these cells. Reinke crystals and paracrystalline inclusions are absent. Gap junctions are common in this system and are notable in the asymmetric nature of the adjacent cytoplasmic components. These findings provide a morphologic correlate to the reported neonatal phase of testosterone production in man. Intermediate forms of Leydig cells are described with "organelle associations" including decreased SER with increased lipid droplets, and decreased SER with prominent cytoplasmic filaments and/or dramatic mitochondrial changes supportive of mitochondrial involution. Cells consistent with immature Leydig cells are also present. The rather impressive diversity in cell morphology present during this time frame of 4 months, slightly past the peak in testosterone production, provides evidence of Leydig cell regression and a continuity of the mature neonatal Leydig cells with the immature Leydig cells of childhood (Prince, 1984). There is also some evidence of cell degeneration. Although the developmental history of Leydig cells has been described for years as biphasic, it is time to view Leydig cell development in man as a triphasic event, fetal, neonatal, and pubertal.

Ultrastructural criteria in evaluating leukemic infiltration in prepubertal testicular biopsies.

The diagnosis of leukemic infiltration in the testis by routine histology is not always possible. During the past 5 years, 46 bilateral testicular biopsies from prepubertal boys were examined by electron microscopy for the purpose of establishing the presence or absence of leukemic infiltration. The ultrastructure of the cells of acute lymphoblastic leukemia is described from the positive cases and compared with cases from the literature. Variations in ultrastructure between the T-cell, B-cell, and "null cell" types are discussed. The ultrastructure of the normal prepubertal cellular elements, which include immature Leydig cells, primitive fibroblastic cells (intertubular), and attenuated peritubular fibroblasts, is described. Ultrastructural criteria are summarized that enable a definitive evaluation of cases that are equivocal by histologic examination.

Ultrastructure of immature Leydig cells in the human prepubertal testis.

The cellularity of the human prepubertal testicular interstitium has not been well studied at the ultrastructural level. In this study, testicular biopsies were obtained from 35 boys aged three to nine years and examined by electron microscopy to clarify and quantitate the cell types present during the prepubertal period. The prepubertal testicular interstitium is found to consist of immature Leydig cells (9%), primitive fibroblastic cells (63%) (intertubular in location), and attenuated peritubular fibroblasts (28%). The primitive fibroblastic cells and peritubular fibroblasts appear closely related, being distinguished mainly by shape and location. The immature Leydig cell type contrasts with the fibroblastic cell types by exhibiting an irregular nucleus with relatively little heterochromatin. The most impressive cytoplasmic feature is the moderate to extensive development of smooth endoplasmic reticulum in the form of anastamosing tubules. In contrast, the rough endoplasmic reticulum is not well developed. Other cytoplasmic characteristics are the highly developed Golgi elements and occasional lipid droplets and lysosomes. Glycogen is also often present and is generally found in those cells that do not contain a well-developed smooth endoplasmic reticulum. The ultrastructure of the immature Leydig cell is compared with that of the mature fetal and adult Leydig cells. Although generally found in small clusters between tubules, these cells are often attenuated and closely associated with the seminiferous tubules. Occasional intermediate cell morphologies suggest a relationship between the primitive fibroblasts and immature Leydig cells. The presence of small cells exhibiting a steroid-producing morphology, classified as immature Leydig cells, in the prepubertal testicular interstitium is an interesting finding and is in accordance with earlier studies on nonhuman mammals. It is unknown whether these cells are remnants of the fetal Leydig cell population or have differentiated neonatally from the primitive fibroblastic cells. It is suggested that the immature Leydig cells are the progenitors of the adult Leydig cell population.

Cultured neonatal rat oligodendrocytes elaborate myelin membrane in the absence of neurons.

We have utilized transmission electron microscopy to study oligodendrocyte-enriched cell cultures established from dissociated neonatal rat cerebra by the method of McCarthy and de Vellis [1980]. Cells were examined after 14 and 26 days in vitro. The overall morphology of the cells from cultures at both time periods was similar and consistent with previous reports of light (immature) oligodendrocyte fine structure. The cells contained an eccentrically located nucleus, prominent Golgi regions, numerous free ribosomes, and microtubules. Large numbers of processes with varying diameter were also observed. There was some indication of cytoplasmic maturation from the younger to the older cultures. The most important feature of the 26-day cultures was the large quantity of intercellular membranes which were shown to be continuous with oligodendrocyte processes. These membranes often exhibited the appearance of "loose myelin" and were therefore not normally compacted. Layers of membrane with the morphologic appearance of compact myelin were observed on an occasional oligodendrocyte perikaryon or process. This finding necessitates a reevaluation of the widely held theory that oligodendrocytes are not able to elaborate myelin in the absence of neurons.

A morphometric analysis of human muscle fibers with relation to fiber types and adaptations to exercise.

Muscle biopsies were obtained from the vastus lateralis of 14 male subjects: 3 long distance runners, 2 world class power lifters and 9 active, although not highly trained, individuals used as controls. The fibers were investigated by electron microscopy and the mitochondrial volume percent, lipid volume percent and Z-line width were analyzed morphometrically. With the combined data a direct correlation was found between mitochondrial volume percent and lipid volume percent, lipid volume percent and Z-line width and mitochondrial volume percent and Z-line width. The muscle fibers were classified as slow-twitch oxidative (SO), fast-twitch-oxidative-glycolytic (FOG) and fast-twitch-glycolytic (FG) based on relationships found in the data and well established properties of muscle fiber types. Although no distinct patterns emerged, a good approximation of fiber type characteristics was obtained, and values for volume percent of central mitochondria, volume percent lipid and Z-line width are reported. The fibers classified as SO were characterized by having wide Z-lines, a high mitochondrial volume percent and high lipid volume percent. The fast-twitch fibers (fibers with narrow Z-lines) were separated into 2 groups, those with high mitochondrial volume percent (FOG) and those with low mitochondrial volume percent (FG). No distinction could be made between the fast-twitch subgroups with regard to Z-line width. The fibers from distance runners differed from those from controls by exhibiting a greater capacity for aerobic activity as evidenced by the increased volume percent of mitochondria and lipid in both slow- and fast-twitch fibers. The high strength, anaerobic activity of the world class power lifters was reflected by the low mitochondrial volume percent of many fast-twitch fibers (FG) and the decreased lipid stores in all fibers.

Ultrastructural aspects of myogenesis found in neoplasms.

The ultrastructure of myogenic cells occurring in neoplasms was investigated and aspects of differentiation (myofilament interactions and organization, and sarcotubular development) were characterized. The stages of muscle differentiation present were extremely similar to those reported during human fetal development prior to innervation. Exceptions, however, being: (1) the presence of fewer multinucleated cells; (2) the general lack of cell elongation and its apparent effect on myofibril orientation; and (3) evidence of a higher number of myofilaments in mononucleated cells. The findings were compared to those reported in normal human fetal development, human myogenic cells in vitro and the literature on mammalian and avian muscle development and discussed with regard to the influence of tension and innervation. The significance of degenerating myogenic cells found in these neoplasms is also discussed.

Muscle fiber types in women athletes and non-athletes.

Muscle biopsies were obtained from the vastus lateralis muscle of 5 female collegiate field hockey players and 5 untrained female students. The fibers were classified histochemically as fast-twitch-oxidative-glycolytic (FOG), fast-twitch-glycolytic (FG) and slow-twitch-oxidative (SO). The fibers were found to be similar to those of males in distribution and histochemical properties, but were smaller. In the women athletes all 3 fiber types were larger than the respective fibers in the controls. Also, the athletes had a much higher percentage of oxidative fibers (SO + FOG), 83% vs. 46%. A direct relationship between fiber size and oxidative activity was observed in fast-twitch fibers, whereas the reverse was found in slow-twitch fibers.

Human muscle fiber types in power lifters, distance runners and untrained subjects.

Muscle biopsies were taken from the vastus lateralis of 12 males: 5 control subjects, 4 power lifters and 3 distance runners. Three fiber "types" were distinguished by comparing serial sections for alkaline myofibrillar adenosine triphosphatase (ATPase) and succinic dehydrogenase (SDH) activities: 1. high ATPase and low SDH; fast-twitch-glycolytic (FG). 2. High ATPase and high SDH; fast-twitch-oxidative-glycolytic (FOG). 3. Low ATPase and high SDH; slow-twitch-oxidative (SO). In some cases the distinction between the FOG and FG classess was not clear and a group termed "transitional" was employed. A variation in percentage of fiber types and fiber area was found among individuals. The percentage of SO fibers varied from 19.6-60.1% within all 3 groups, with a mean of 40.5%. In the control group approximately 75% of the fibers were oxidative (FOG + SO). The major characteristics of the lifters were a decrease in the percentage of FOG fibers and a hypertrophy of FOG and FG fibers. The distance runners had a high percentage of oxidative fibers with few FG fibers. It is suggested that the fast-twitch fibers are mainly involved in the adaptation of muscle to exercise since the percentage of SO fibers varies greatly among individuals within and between the 3 groups studied.