Electrophoretic and immunocytochemical analysis of Hsp72 and Hsp73 expression in heat-stressed mouse testis and epididymis.

OBJECTIVE: One of the most profound events in stressed cells is the synthesis of a highly conserved family of proteins, the 'heat shock proteins' (Hsp). The Hsp70 family is the most diverse, and includes constitutive as well as stress-inducible proteins with overlapping or unique functions in different cell compartments. Elucidation of Hsp70 expression during different stages of spermatogenesis and maturation of germ cells is of particular interest due to their high sensitivity to heat treatment. STUDY DESIGN: Expression of the main isoforms of the Hsp70 family (constitutive Hsp73 and stress-inducible Hsp72) was determined in normal and heat-stressed mouse testes and epididymis from sexually mature (60-day-old) mice during spermatogenesis and maturation of germ cells. Immunocytochemical analysis and one- and two-dimensional gel electrophoresis were used to separate mouse testicular and epididymal proteins from saline extracts, followed by Western blotting. RESULTS: Using a polyclonal anti-Hsp70 antibody that recognizes both isoforms, inducible Hsp72 expression, was demonstrated immunocytochemically only in heat-stressed tissues, while a high level of constitutive Hsp73 isoform expression was found in both normal and heat-stressed mouse male reproductive tissues. Morphological studies have shown that round and elongated spermatids in the testes, as well as all segments of the epididymis, are most sensitive to heat stress. In the epididymis, the reaction was localized in different cell compartments. CONCLUSION: In heat stress conditions, Hsp73 is mobilized to prevent apoptosis in the testes and epididymis, and assists Hsp72 in the repair of stress-altered protein conformations.

Role of FGF1, FGF2 and FGF7 in the development of the pancreas from control and streptozotocin-treated hamsters.

Although progress has been made with respect to the growth and transcription factors implicated in pancreatic development, many questions remain unsolved. It has been established that during embryonic life, both endocrine and acinar cells are derived from pancreatic epithelial precursor cells. Growth factors control the proliferation of precursor cells and their ability to differentiate into mature cells, both in pre-natal and in early post-natal life. Pancreatic development during the early post-natal period is an area of great interest for many scientists. In this study we have examined the structure characteristics, functional and proliferative activity of control and diabetic hamster pancreatic ductal, exocrine and beta cells, following treatment with FGFs 1, 2 and 7 in vitro. Light and electron microscopic studies indicated active synthetic processes in these cells under the influence of the investigated FGFs. In our experimental model of diabetes, the labelling index of the cells was significantly higher than in corresponding control groups of hamsters. We established that FGF2 at a concentration of 10 ng/l was responsible for the most prominent effect on ductal cells and beta cells in the diabetic groups. FGF1 at a concentration of 10 ng/l displayed the highest stimulatory effect on exocrine cells in the diabetic groups at post-natal day 10. Taken together these data strongly suggest that FGF1 and FGF2 induce proliferation of pancreatic epithelial cells during the early post-natal period whereas FGF7 is not strictly specific for pancreatic cell proliferation.

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.

Effect of orchiopexy on human testicular ultrastructure.

The effect of orchiopexy performed before puberty on the testicular ultrastructure in sexually mature infertile men have been levels, as well as an analysis of spermogram and karyotype was carried out. It was found an elevation of FSH and LH, while T levels were decreased. Disturbances in the ultrastructure of all testicular cell types-germ, Sertoli and Leydig cells are related with the smooth and rough endoplasmic reticulum and mitochondria as well. It is concluded that the application of orchiopexy in 8-10 years old cryptorchid boys have no curative effect on the descended testes.

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.

Testicular ultrastructure in infertile men.

The review shows typical ultrastructural alterations of germ, Sertoli, and Leydig cells in infertile men. Regardless of the cause of infertility, the disruption of the spermatogenic process usually occurs in the pachytene stage of meiotic prophase and in the stages of early spermatid maturation. The disturbances affect the cytoplasm more than the nucleus, and the synaptonemal complexes have shown significant stability even in the severely injured testes. An acrosome formation is found to be open to injury in more advanced germ cells during spermatid maturation. The manner of reaction of the Sertoli cells under different pathological conditions depends on the presence and degree of maturation of the neighboring germ cells. The appearance of immature Sertoli cells is accompanied by the loss of germ cells more advanced in their differentiation. In most pathologically altered testes, mature Sertoli cells reveal a universal manner of reaction of cell organelles. Leydig cell ultrastructure fluctuates considerably, and the alterations predominantly affect the sites of steroid synthesis, in spite of disease specificity. It becomes clear that a complex estimation of a real testicular state requires the application of new techniques as well as recognition of local control mechanisms. This will provide evidence toward elucidation of male infertility.

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.

Histoautoradiographic studies of 3H-thymidine labelled Erysipelothrix rhusiopathiae introduced into albino mice.

S-forms of E. rhusiopathiae were cultivated in a 3H-thimidine-containing medium. A suspension washed three times in succession was intravenously injected into albino mice. At certain intervals liver, spleen and kidney were examinated by routine autoradiogrphy. Intact labelled bacteria were found as early as 5 min after administration in the blood capillaries, around cells of the RES and in some nuclei of hepatocytes. Later they are detected in the cytoplasm of micro- and macrophages, around megacaryocytes and in some of their nuclei. After 6 h the labelled population sharply decreases due to a very active reproduction and mortality of the initial population. The localisation of bacteria in liver nuclei and the spreading of S-forms of E. rhusiopathiae in comparison with the avirulent L-forms therefrom are discussed.

Histoautoradiographic studies on the fate of 3H-thymidine labelled L-forms of E. rhusiopathiae in albino mice.

L-forms of Erysipelotrix rhusiopathiae labelled with 3H-thymidine were intravenously administered to albino mice. Autoradiographic studies of the liver, kidney and spleen were undertaken at periods ranging from 2 minutes to 15 days. On the second minute following the administration of the radioactive material whole labelled microorganisms and chains of silver grains were recovered in the examined organs. Up to the 15th minute labels were observed also in the cells of the RES. Following the 30th minute the silver grains were positioned at a characteristic site in the Golgi region of the hepatocytes. At the same time in the kidney they were localized in the glomerular space and in the lumen of the renal tubules, whereas in the spleen - mainly around the megakaryocytes. By the 15th day labelling gradually diminished, single silver grains being found over some nuclei of megakaryocytes, liver and kidney parenchymal cells. The present study throws light over some aspects of the interrelationship between the micro- and macroorganism concerning the mechanisms of desintegration, elimination and the uptaking of labelled microbial DNA.