Accelerated maturation of primate testis by xenografting into mice

Testicular maturation and sperm production throughout the life of the male form the basis of male fertility. It is difficult to elucidate the intricate processes controlling testicular maturation and spermatogenesis in primates in vivo due to the long time span required for sexual maturation and also to the lack of accessible in vitro or in vivo models of primate spermatogenesis. Ectopic xenografting of neonatal testis tissue into mice provides an accessible model to study and manipulate the propagation and differentiation of male germ cells from immature donor animals. However, it was not clear whether this approach would be applicable to slowly maturing primates. Here we report that grafting of testis tissue from immature rhesus monkeys (Macaca mulatta) into host mice resulted in the acceleration of testicular maturation and production of fertilization-competent sperm in testis xenografts. The system reported here provides a powerful, practical approach to study timing and control of testicular maturation and regulation of primate spermatogenesis without the necessity for experimentation in primates. This approach could potentially be applied to produce fertile sperm from sexually immature individuals of rare or valuable primate species or from prepubertal boys undergoing sterilizing therapy for cancer.     

Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis

The physiological apoptosis that occurs in immature testis appears to be necessary for the maturation of this tissue. Thus, inhibition of the early apoptotic wave associated with the first round of spermatogenesis is followed by accumulation of spermatogonia and infertility later in life. To identify the cell types undergoing apoptosis in immature rat testis and to characterize the relationship between this apoptosis and progression of the first wave of spermatogenesis, sequential viable segments of seminiferous tubules from 8-, 18-, and 26-day-old rats were examined under a phase-contrast microscope. One novel observation was the existence of pronounced stage-specificity during the peak of apoptosis at the very early postnatal ages of 18 and 26 days. Increased apoptosis of pachytene spermatocytes in stages VII-VIII was the major feature that distinguished immature spermatogenesis from the corresponding adult process. The frequency of apoptosis among type A spermatogonia in immature stages IX-I was also elevated in comparison to the corresponding mature stages. The age-related peak of apoptosis was mediated by caspase 3; furthermore, stage-dependent expression of Bax in midpachytene spermatocytes was observed in the 18- and 26-day-old testis. These observations suggest that this Bax-regulated, caspase 3-mediated, increased apoptosis of midpachytene spermatocytes during the first wave of immature spermatogenesis represents a major difference in comparison to apoptosis occurring in the mature testis, and it may play an important regulatory role in establishing spermatogenesis in the rat testis.     

Interleukin-6 expression during normal maturation of the mouse testis

In this study, we examined the cellular origin and the expression levels of interleukin-6 (IL-6) during normal maturation of mouse testis. The levels of IL-6 (protein and mRNA) were higher in testicular homogenates of sexually immature than mature mice. Immunohistochemical staining of testicular tissues of sexually immature and adult mice show that testicular germ cells, at different stages of differentiation, Leydig cells/interstitial cells and peritubular cells express IL-6. Our results demonstrate, for the first time, overexpression of IL-6 in testicular tissues of immature mice, as compared to mature mice, as well as the expression of IL-6 in germ cells of testicular tissues of adult and sexually immature mice. Thus, our results may indicate the involvement of the endocrine system (gonadotropins and testosterone) in the regulation of IL-6, which is involved in the regulation of testicular development, functions and spermatogenesis.     

Luteinizing hormone receptor-mediated effects on initiation of spermatogenesis in gonadotropin-deficient (hpg) mice are replicated by testosterone

Testosterone (T) is an absolute requirement for spermatogenesis and is supplied by mature Leydig cells stimulated by LH. We previously showed in gonadotropin-deficient hpg mice that T alone initiates qualitatively complete spermatogenesis bypassing LH-dependent Leydig cell maturation and steroidogenesis. However, because maximal T effects do not restore testis weight or germ cell number to wild-type control levels, additional Leydig cell factors may be involved. We therefore examined 1). whether chronic hCG administration to restore Leydig cell maturation and steroidogenesis can restore quantitatively normal spermatogenesis and testis development and 2). whether nonandrogenic Leydig cell products are required to initiate spermatogenesis. Weanling hpg mice were administered hCG (0.1-100 IU i.p. injection three times weekly) or T (1-cm subdermal Silastic implant) for 6 weeks, after which stereological estimates of germinal cell populations, serum and testicular T content, and testis weight were evaluated. Human CG stimulated Leydig cell maturation and normalized testicular T content compared with T treatment where Leydig cells remained immature and inactive. The maximal hCG-induced increases in testis weight and serum T concentrations were similar to those for T treatment and produced complete spermatogenesis characterized by mature, basally located Sertoli cells (SCs) with tripartite nucleoli, condensed haploid sperm, and lumen development. Compared with T treatment, hCG increased spermatogonial numbers, but both hCG and T had similar effects on numbers of spermatocytes and round and elongated spermatids per testis as well as per SC. Nevertheless, testis weight and germ cell numbers per testis and per SC remained well below phenotypically normal controls, confirming the involvement of non-Leydig cell factors such as FSH for quantitative normalization of spermatogenesis. We conclude that hCG stimulation of Leydig cell maturation and steroidogenesis is not required, and that T alone mostly replicates the effects of hCG, to initiate spermatogenesis. Because T is both necessary and sufficient for initiation of spermatogenesis, it is likely that T is the main Leydig cell secretory product involved and that additional LH-dependent Leydig cell factors are not essential for induction of murine spermatogenesis.     

Isolation and biochemical characterization of nuclei from immature and mature guinea pig granulocytes

Intact nuclei were isolated in high yield from enriched fractions of immature and mature guinea pig granulocytic leukocytes. These nuclei were used to determine whether any changes in synthesis and content of nuclear proteins accompany the striking increase in chromatin condensation and the nuclear lobation which occur during granulocyte maturation. The results indicate that the synthesis of nuclear proteins and the nuclear RNA content decrease markedly during granulocyte maturation. The incorporation of l-[U-¹⁴C]leucine into the acid-soluble histone-rich fraction of chromatin from immature cells is about 25 times that of mature cells, and the incorporation into the acid-insoluble, nonhistone proteins of chromatin from immature cells is about 6 times that of mature cells. It appears that there is very little quantitative change with respect to the protein components of nuclei from immature and mature granulocytic leukocytes. No significant differences in the amounts of histone, nonhistone protein, or phosphoprotein between nuclei of immature and mature granulocytes could be detected. No major differences in gel electrophoretic patterns of histones or nonhistone proteins could be detected. The fact that the amount of the chromatin proteins remains relatively constant during cell maturation in spite of the pronounced decrease in the rate of synthesis suggests that the rate of turnover of these proteins decreases significantly as the maturation of granulocytic leukocytes proceeds.     

Chromatin maturation depends on continued DNA-replication

The structure of [3H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [3H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. We conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation.     

Differential expression of bcl-2 and bcl-x during chicken spermatogenesis

We report the isolation and characterization of a chicken testis bcl-x_L cDNA coding for a long bcl-x protein with a hydrophobic tail, and the expression of bcl-2 and bcl-x during chicken spermatogenesis. Bcl-2 is highly expressed in embryonic and immature testes enriched in spermatogonia and barely detectable in mature testes, where most of the cells are meiotic and postmeiotic. Bcl-x is expressed in both mature and immature testes, but in a lesser amount in mature testes. Differential expression of bcl-2 and bcl-x during spermatogenesis is consistent with the reported different susceptibility to apoptosis of spermatogonia, and meiotic and postmeiotic cells. Mol. Reprod. Dev. 47:26–29, 1997. © 1997 Wiley-Liss, Inc.     

Over-expression of IL-18, ICE and IL-18 R in testicular tissue from sexually immature as compared to mature mice

In this study we examined the cellular origin and the expression levels of interleukin-18 (IL-18), IL-18 receptor (IL-18R) and IL-1beta-converting enzyme (ICE), which activates pro-IL-18, during normal maturation of murine testis. The levels of IL-18, IL-18R and ICE were significantly higher in testicular tissues and homogenates (but not in the spleen or liver) from sexually immature than mature mice. Immunohistochemical staining of testicular tissues from sexually immature and mature mice shows that testicular germ cells and Leydig cells/interstitial cells express higher levels of IL-18, as compared to other testicular cells. Peritubular cells of sexually immature and mature mice also expressed IL-18. Our results demonstrate, for the first time, over-expression of the IL-18 family in testicular tissues of sexually immature mice, as compared to mature mice, as well as the expression of IL-18 in the different stages of differentiation of testicular germ cells. Thus, our results may indicate involvement of the endocrine system (gonadotropins and testosterone) in the regulation of the testicular IL-18 family, which could be involved in the regulation of testicular functions, development and spermatogenesis under physiological conditions.