Leukaemia inhibitory factor stimulates proliferation of prospermatogonial stem cells.

The effect of leukaemia inhibitory factor (LIF) on the proliferation of prospermatogonial stem cells was tested in vitro. Pieces of 3-day-old rat testis were cultured in the presence of a range of doses of LIF alone or in combination with 1 ng mL(-1) seminiferous growth factor (SGF). Stimulation of the proliferative activity of quiescent prospermatogonia was detected immunocytochemically with a cell proliferation kit. After 24 h culture, LIF significantly increased the percentage of labelled prospermatogonia, with the peak of activity at 20 pg mL(-1). The combination of LIF and SGF resulted in a decrease in DNA synthetic activity of the germ cells. Hence, LIF and SGF play a role in local regulation at the onset of spermatogenesis in the rat testis.

Differential effects of prepubertal rat Sertoli cell secreted proteins on somatic testicular and nontesticular cells.

There is little information on the mitogenic and immunoregulatory activities of proteins, secreted by prepubertal Sertoli cells during the stage of meiosis initiation and before creation of the blood-testis barrier. We have previously demonstrated dose-dependent and age-related stimulation of BALB/c 3T3 fibroblasts and quiescent rat prespermatogonia (Kancheva et al., 1990) as well as inhibition of natural killer cell activity of mice, guinea pigs and human lymphocytes (Nikolova et al., 1992) by Sertoli cell-conditioned medium derived from 12-day-old rats. In the current study, using splenic lymphocytes stimulated by PHA, LPS and Con A, we have shown a dose-dependent inhibition of T and B lymphocyte proliferation by prepubertal Sertoli cell-secreted proteins (pSCSP). These results suggest that by the time the blood-testis barrier had been formed, Sertoli cell in rat testis had already synthesized immunoregulatory proteins. In addition we have found that pSCSP stimulate the proliferation of TM3 Leydig but not TM4 Sertoli cells. The differential effect of pSCSP is an expression of the different balance between growth factors secreted by Sertoli cells, which in turn is dependent on the requirements of the cell types at each stage of testicular development.

The perinuclear matrix as a structural element of the mouse sperm nucleus.

The mouse sperm nucleus, after the removal of protamines and DNA, consisted of a skeletal structure that conformed to the original nuclear shape. Sperm were extracted with 1% SDS, and the isolated nuclei, along with the enveloping perinuclear theca, were incubated in 25 mM dithiothreitol, and exposed to different reagents in an effort to displace the protamines, P1 and P2. Protamines, labeled with [3H]arginine, were displaced from the nucleus by CaCl2.MgCl2, but only partially by anionic detergents, monovalent cations, and polyvalent anions. Displacement of P1 and P2 was achieved by digesting the nuclei with DNase I and simultaneously extracting with CaCl2.MgCl2 (3:2; mol:mol) in stepwise increments of 125, 150, 175, 200, and 250 mM. Protamine displacement was concentration-dependent, occurring with an EC50 of approximately 205 mM and with maximal displacement at approximately 250 mM CaCl2.MgCl2. The nucleus was reduced to a skeletal structure consisting of the perinuclear theca and an internal network of transverse fibers. The evidence was consistent with the former being derived from the perforatorium and postacrosomal nuclear sheath (both cytoplasmic structures), whereas the fibers were most likely of nuclear origin. By SDS-PAGE and isoelectric focusing (IEF), perinuclear matrices consisted of greater than or equal to 230 protein spots, with M(r)s in the range of 70,000 to 8000 and pIs of greater than or equal to 7.5 to approximately 4.7, respectively. Monoclonal antibodies prepared against perinuclear matrices bound to specific proteins on IEF immunoblots and, based on light and electron microscopic observations, to discrete domains of the sperm perinuclear theca and nucleus.

Species-specific effect of proteins secreted by cultured pre-pubertal rat Sertoli cells on natural killer cell activity.

The effect of proteins secreted by cultured pre-pubertal rat Sertoli cells (pSCP) on natural killer (NK) cell activity of rat, mice and guinea pig splenocytes and human peripheral blood lymphocytes was estimated. The results indicate that pSCP inhibited, in a dose-dependent manner, NK cell activity of mice, guinea pig and human lymphocytes but did not suppress lysis of YAC-1 target cells by rat NK cells. Species-specific differences in the effect of pSCP on NK cell activity probably result from differences in the binding of proteins within the effector cells. These data indicate that in the pre-pubertal period of gonadal development immature Sertoli cells synthesize a factor/s which might contribute to the maintenance of specific testis immunological environment.

Prepubertal rat Sertoli cells secrete a mitogenic factor(s) that stimulates germ and somatic cell proliferation.

Sertoli cells were isolated from prepubertal 6- and 12-day-old rats. The Sertoli cell-conditioned media (SCCM-6 and SCCM-12) can markedly stimulate the proliferation of somatic cells and quiescent rat prespermatogonia in a dose-dependent and an age-related manner. SCCM-12 stimulated cell proliferation of BALB/c 3T3 fibroblasts up to 7-fold over control values, but did not stimulate to the same degree the germ cell mitotic activity. SCCM-6 stimulated proliferation of prespermatogonia up to 5-10-fold over controls. The mitogenic factor(s) in SCCM-6 appears to be more specific to prespermatogonia than to somatic cells which is consistent with the in vivo stimulation of mitosis in germ cells 5-6 days after birth and with the action of 'mitosis inducing substance'. The mitogenic factor(s) appears to be protein with a molecular weight over 8000 and sensitive to heat and trypsin treatment. These results suggest that the different mitogenicity of prepubertal rat SCCM on germ and somatic cells may be due to secretion of multiple mitogens by Sertoli cells in an age-dependent manner.

Ultrastructural alterations in mouse spermatogenic cells after treatment with anticancer drug biocarbazine.

Single doses of anticancer drug biocarbazine (BC)--DTIC synonym--were injected intraperitoneally at 50 and 200 mg/kg body weight to sexually mature BALB/c mice. Among other features previously reported (Martinova et al. 1989) BC causes ultrastructural alterations in spermatogonia and spermatocytes significantly expressed after administration of higher dose. In addition BC induces some defects in acrosome formation of early spermatids in Golgi and cap phase, while spermatids in later stages of maturation showed marked resistance to BC treatment.

An immunocytochemical study of the proliferating cell nuclear matrix antigen p125/6.5 during rat spermatogenesis.

In previous studies of proliferating mammalian cells a p125/6.5 nuclear matrix antigen displaying a marked increase in mitotic cells has been identified. This antigen was investigated by immunocytochemistry of cryosections of testes at different stages of postnatal development: newborn, 20 days after birth and sexually mature rats. In Sertoli cells, the distribution of the p125/6.5 antigen parallels [3H]thymidine incorporation: present in newborn and absent in sexually mature testes. The p125/6.5 antigen is present also in some prespermatogonia of the newborn rat testis, which do not incorporate [3H]thymidine. At later stages of development, the p125/6.5 antigen is present also in first meiotic prophase spermatocytes displaying an extrachromosomal nucleoplasmic distribution, while absent in spermatids and spermatozoa. These results show that the p125/6.5 antigen increases not only during mitosis, but also during meiosis. They suggest further that this antigen is characteristic of both proliferating cells and cells (prespermatogonia) committed to proliferation.

Early effect of the anticancer drug biocarbazin (DTIC synonym) on mice spermatogenesis.

Biocarbazin (DTIC synonym) is an anticancer drug acting as a purine analogue, as an alkylating agent, as a SH-group blocker. Biocarbazin administration in doses 50 and 200 mg/kg of body weight to adult male BALB/c mice suppresses the process of spermatogenesis. Seminiferous tubule of the testis displayed dose-dependent histological alterations manifested with decrease of mitotically dividing cells and increase in the number of multinucleated spermatocytes and spermatids. RNA synthetic activity studied by means of autoradiography showed a tendency for a reduction in the spermatogonia and pachytene spermatocytes.