Mechanisms for the testicular hypertrophy which follows hemicastration.

The hormonal and testicular effects of hemicastration have been examined using rodent models. When rats were hemicastrated at 5 days of age, significant testicular hypertrophy was noted within 5 days. Hypertrophy decreased as the age at hemicastration approached 20 days and did not occur in rats 45 days of age or older. The changes in testicular weight were associated with significant (P less than 0.001) increases in serum FSH values from 10-20 days, but no significant alterations in the intratesticular concentration of testosterone occurred. Hemicastration also caused significant hypertrophy in testes depleted of germ cells. This was reflected by a substantial increase in testicular protein and DNA when compared to intact controls at 10, 15, and 20 days of age; however, the concentration of protein/microgram DNA was increased only at 15 days. These data indicate that 1) changes in serum FSH and not intratesticular testosterone are associated with the testicular hypertrophy which follows unilateral castration of immature animals; 2, a significant proportion of the hypertrophy can be attributed to non-germinal cells, the Sertoli cell being the prime candidate;3) the increase in testicular weight is primarily the result of an increase in cell number.

Cryptorchid rhesus macaques: long term studies on changes in gonadotropins and gonadal steroids.

We studied the relationship of testosterone (T), dihydrotestosterone (DHT), and 27 beta-estradiol (E2) to FSH and LH in the systemic circulation of six rhesus macaques with surgically induced cryptorchidism at selected times over 420 days. We measured these hormones by RIA and compared their concentrations with those of four sham-operated controls. Midway through the experimental procedure the animals were electroejaculated, and on day 425 the testes were removed and analyzed histologically. The cryptorchid monkeys did not have sperm in their ejaculates, and treatment adversely affected spermatogenesis. For the first 30 days after treatment none of the hormones differed significantly between cryptorchid monkeys and controls. Gonadotropins in three castrated males, however, gradually rose to postcastration levels within approximately 2 weeks post operation. After castration, all three steroids declined significantly within 24 h. Beginning on days 60, 150, 215, 320, and 420 post operation the animals were bled for 5 days on a diurnal regimen. The steroids as well as LH concentrations varied diurnally. The concentration of FSH never showed diurnal variation in any of the groups at any of the periods studied. Concentrations of T and LH did not differ significantly between control and cryptorchid groups at any time. With longer time periods (beginning 215 days after cryptorchidism had been induced), DHT in cryptorchid monkeys was significantly higher than in controls (P < 0.01). Although E2 was significantly lower in cryptorchid monkeys on days 60 and 150 post operation, this difference disappeared with time. Changes in steroid concentrations were not associated with the elevations in FSH that occurred in cryptorchid monkeys by days 60 and 420. Therefore, we assumed that they were independent phenomena. Significant FSH elevations in cryptorchid monkeys occurred only in the fall of the year. Testicular homogenates from cryptorchid and control monkeys produced similar quantities of T, DHT, and androstenedione in vitro. Very little E2 or estrone was found. Although significant amounts of progesterone were quantified in the incubation media of control testes, little or no progesterone was found in media from cryptorchid testes. Similar results were obtained when these steroids were measured in plasma collected from the testicular veins. In testicular venous plasma, 20 alpha-dihydroprogesterone concentrations were not elevated after cryptorchidism. These data suggest that there is similar negative feedback control of gonadotropins in crytorchid and control rhesus monkeys. The absence of dramatic differences in systemic concentrations of FSH between the two groups suggests that the seminiferous tubule does not play a major role in the negative feedback control of gonadotropins in this species or that the tubular component of this control system is not perturbed by the cryptorchid condition...

An FSH and prolactin-secreting pituitary tumor: pituitary dynamics and testicular histology.

A man with normal-sizes testes and a chromophobe adenoma of the pituitary was found to have elevated plasma levels of FSH and prolactin and severe deficiencies of other trophic hormones. Plasma FSH values doubled after administration of LH-releasing hormone but were not suppressed by exogenous testosterone. Prolactin concentrations were increased by TRH and were suppressed by L-dopa. Testicular histology revealed sparse and apparently inactive Leydig cells. Seminiferous tubules and Sertoli cells were well preserved, but there were few late spermatids.

Spermatogonial intercellular bridges in whole-mounted seminiferous tubules from normal and irradiated rodent testes.

Whole-mounted seminiferous tubules from normal and irradiated rodent testes were examined by light microscopy. These studies reveal the presence of intercellular bridges in all classes of spermatogonia except for the new As stem cells. It was demonstrated that As stem cells divide to produce new As spermatogonia or paired daughter cells that are united by a cytoplasmic bridge. Evidence was given that all subsequent progeny of these paired A's up to and including the production of type B spermatogonia remain linked by cytoplasmic bridges in increasingly larger and more complex syncytial networks. It is proposed that the intercellular bridges mediate both differentiation and degeneration of spermatogonia. The maintenance of synchronous development within cohorts of spermatogonia is attributed to the bridges. Moreover, the fact that spermatogonia in both normal and irradiated testes degenerate in clusters is determined by the presence of intercellular bridges. Lastly, the integrity of the bridges appears essential for normal germ cell development.

The morphology and kinetics of spermatogonial degeneration in normal adult rats: an analysis using a simplified classification of the germinal epithelium.

The phenomena of spermatogonial degeneration have been studied in normal adult rat testes using a simplified classification of the germinal epthelium based upon the six types of differentiating spermatogonia. The following features distinguished this from schemes based on acrosome development. Rather than 14 states of unequal duration, there are only six stages, five of which are the same length. The classification starts at the beginning of spermatogenesis with A1 spermatogonia rather than at the onset of spermiogenesis. The classification is derived from acutal biological events in spermatogenesis, namely generation times of spermatogonia, rather than upon arbitrary events in acrosome development. Most importantly, this new classification can be used with most types of preparations and in most experimental conditions. Examination of tubular whole mounts reveals that degeneration preferentially occurs in types A2 and A3 and to a lesser extent A4 spermatogonia, and is rarely seen in generations of A1, In or B cells. Deterioration is first manifested in clusters of cells joined by the intercellular bridges as they complete DNA synthesis and enter the 2 phase of cell cycle. It is characterized by a denser staining of the nuclear membrane, coalescence of chromatin into several pyknotic bodies, and eventual extrusion of the nuclear mass, leaving a cytoplasmic ghost. The sequential steps in degeneration may often be traced from one end of a synctial chain to the other, suggesting that the process may start with just one cell and then spread via intercellular bridges to involve all spermatogonia within the clone. Quanitatively, degeneration is a relatively constant feature of spermatogonial development. Only 25% of the theoretically possible number of pre-leptotene spermatocytes are produced from th original population of A1 spermatogonia; most of this loss is incurred during the maturation of A2 and A3 generations. While the reason for spermatogonial degeneration in the normal generminal epithelium remain obscure, it is proposed that the numerical ratio of A spermatogonia to Sertoli cells may be a significant limiting factor.

Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules, I. The normal testes.

The spermatogonial populations in ten normal adult mice were analyzed using whole mounted seminiferous tubules. The undifferentiated A spermatogonia as well as the six generations of differentiating spermatogonia were clearly identifiable on whole mounts. Description plus quantitation of these cell types revealed that they behaved in essentially the same manner as their counterparts in the rat. Single undifferentiated A cells were classified as type As stem cell spermatogonia. They were distributed throughout the seminiferous epithelium, and by periodic mitoses, maintained their stock and furnished cells which would eventually differentiate. Although initially resembling the As spermatogonia, the progeny which were destined to differentiate were classified as type Aal spermatogonia because they were linked by cytoplasmic bridges, and because they usually underwent one or more synchronous mitotic divisions to form short chains of aligned cells. Ultimately, division of Aal cells were no longer seen, and the cells appeared to gradually acquire the typical morphological characteristics of A1 spermatogonia; these continued to differentiate according to the well-established pattern. It was concluded that the cyclic production of cohorts of A1 cells in this manner would ensure a continual supply of spermatogonia for differentiation.

Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules. II. The irradiated testes.

In adult male mice exposed to 300 R X-irradiation, the spermatogonial population was selectively killed except for the radioresistant type As stem cells. Type A spermatogonia were minimal two days after irradiation, when only 20% of the control population was present in stage 5-6; these were predominately single and paired undifferentiated cells. When multiple injections of 3HTdR were given between 2 and 3.5 days post-irradiation, 90-95% of these survivors in stages 4-6 became labeled. Enhanced proliferation of these stem cells, and at times when they were normally quiescent, led to restoration of all classes of spermatogonia by 11 days after irradiation. Several autoradiographic studies were undertaken to better characterize the radioresistant cells. In mice given single or multiple injections of 3HTdR prior to irradiation, there was appreciable retention of label by those type As spermatogonia that had originally incorporated 3HTdR in stages 2-4. This labeling pattern was identical to that of the long-cycling As stem cells in nonirradiated testes. Since the long-cycling As stem cells are thought to be characterized by a prolonged G1 or "A-phase" which is known to be a highly radioresistant portion of the cell cycle, it was clear why these cells could preferentially survive irradiation doses that killed other spermatogonial types. It was proposed that following germ cell depletion, as after irradiation injury, the long-cycling As survivors could be prematurely triggered from A-phase into DNA synthesis, thereby, initiating restoration of the germ cell population.

Morphological profiles of cryptorchid XXY mouse testes.

Two mice with an XXY karyotype and cryptorchid testes appeared spontaneously in a colony. The animals were H-Y antigen-positive, and had elevated serum levels of follicle-stimulating (FSH) and luteinizing (LH) hormones. Testes of the affected mice were atrophic, containing a few solid seminiferous cords surrounded by vast amounts of compact interstitial material. The cords were delimited by a broad tunica propria in which the basal lamina was irregularly thickened and stratified into a number of alternating dense and less dense layers. Most sex cords were populated by mature Sertoli cells and small pleomorphic elements resembling monocytic-derived macrophages. Within some cords, the macrophages aggregated into a central mass with which identifiable Sertoli cells and (PAS) periodic acid Schiff-positive fragments of basal lamina were associated. In more severely damaged cords, the basal lamina and peripheral carpet of Sertoli cells were totally missing. Such cords were populated only the the central macrophages with fragments of basal lamina and degenerating Sertoli cells. Finally, a few collapsed remnants of cords contained compact nodules of macrophages surrounded by what appeared to be the outer part of the tunica propria. The interstitial area, as well as the outer walls of the seminiferous cords were also heavily infiltrated by macrophages. Overall, the morphological picture was one of severe immunological injury. We do not know what role, if any, the genetic constitution and/or intra-abdominal environment may play in the expression of these bizarre pathologies. However, such severe changes have not been reported in either Klinefelter's syndrome or the undescended testes of any human or subprimate species.

Do spermatogonial stem cells have a circadian rhythm?

Mitotic index was determined in whole mounts of segments of seminiferous tubules of (101 X C3Hf)F1 male mice at 3 hr intervals from 18.00 to 06.00 hours, and at hourly intervals from 08.00 to 16.00 hours. The highest frequency of metaphase-anaphase figures occurred at 10.00 and 11.00 hours, but was not significantly higher than for other times. Injection of 25 mu Ci 3H-TdR per mouse, followed 24 hr later by exposure to 300 rad X-rays and killing 207 hr after labelling was used to test for circadian rhythm in DNA synthetic activity of the long-cycling As spermatogonia. No significant effect of time of day was observed. Likewise, the number of undifferentiated spermatogonia scored 183 hr after 300 rad showed no effect of time of day. The testis therefore appears to have no circadian rhythm in mitotic activity. Stage of the cycle of the seminiferous epithelium, however, showed a significant effect on mitotic index of As spermatogonia and on DNA synthetic activity of undifferentiated spermatogonia. These data are compared with those for other organisms and tissues in respect to which properties of stem cells are general for all organisms and tissues and which are specific for spermatogonia.