Globozoospermia in mice lacking the casein kinase II alpha' catalytic subunit.

Protein kinase casein kinase II (Ck2) is a cyclic-AMP and calcium-independent serine-threonine kinase that is composed of two catalytic subunits (alpha and alpha') and two regulatory beta-subunits. Ck2 is not a casein kinase in vivo, but over 100 substrates are known. The highly conserved amino acid sequences of its subunits and their broad expression suggest that Ck2 may have a fundamental role in cell function. Ck2 has been implicated in DNA replication, regulation of basal and inducible transcription, translation and control of metabolism. The Ck2alpha and Ck2alpha' isoforms (products of the genes Csnk2a1 and Csnk2a2, respectively) are highly homologous, but the reason for their redundancy and evolutionary conservation is unknown. We find here that Csnk2a2 is preferentially expressed in late stages of spermatogenesis, and male mice in which Csnk2a2 has been disrupted are infertile, with oligospermia and globozoospermia ('round-headed' spermatozoa). This is the first demonstration of a unique role for a Ck2 isoform in development. The primary spermatogenic defect in Csnk2a2-/- testis is a specific abnormality of anterior head shaping of elongating spermatids; this is the first defined gene that regulates sperm head morphogenesis. As the germ cells differentiate, they are capable of undergoing chromatin condensation, although many abnormal cells are deleted through apoptosis or Sertoli cell phagocytosis. The few that survive to populate the epididymis exhibit head abnormalities similar to those described in human globozoospermia, thus Csnk2a2 may be a candidate gene for these inherited syndromes.

Testicular degeneration in Bclw-deficient mice.

To identify genes required for mammalian spermatogenesis, we screened lines of mutant mice created using a retroviral gene-trap system for male infertility. Homozygous ROSA41 male mice exhibit sterility associated with progressive testicular degeneration. Germ-cell defects are first observed at 19 days post-natal (p19). Spermatogenesis is blocked during late spermiogenesis in young adults. Gradual depletion of all stages of germ cells results in a Sertoli-cell-only phenotype by approximately six months of age. Subsequently, almost all Sertoli cells are lost from the seminiferous tubules and the Leydig cell population is reduced. Molecular analysis indicates that the gene mutated is Bclw, a death-protecting member of the Bcl2 family. The mutant allele of Bclw in ROSA41 does not produce a Bclw polypeptide. Expression of Bclw in the testis appears to be restricted to elongating spermatids and Sertoli cells. Potential roles for Bclw in testicular function are discussed.

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice.

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.

Germ cell transplantation and the study of testicular function.

Spermatogonial stem cell transplantation is a novel technique in which donor testicular cells are transferred into recipient testes. A population of germ cells from a transgenic or mutant donor is introduced into the seminiferous tubules of recipient testes by microinjection. Following injections, spermatogonial stem cells can colonize the recipient testis, initiate spermatogenesis and produce sperm capable of fertilization. This technique will allow scientists to: (1) investigate fundamental aspects of spermatogenesis; (2) provide a method to regenerate spermatogenesis in infertile individuals; and (3) genetically manipulate spermatogonial stem cells to develop transgenic animals.

Presence and localization of a 30-kDa basic fibroblast growth factor-like protein in rodent testes.

We have used a recently characterized rabbit antiserum against basic fibroblast growth factor (bFGF), which recognizes various forms of bFGF, to examine the presence and localization of bFGF in the testes of adult rats and mice and the 5-day-old rat. In Western blots of testicular homogenates of adult rats and mice and immature rats, immunoreactive single bands at approximately 30 kDa were detected. Immunocytochemistry revealed specific staining restricted to the tubular compartment. In 5-day-old rat testes, prespermatogonia were immunoreactive. The cytoplasm of pachytene spermatocytes was heavily stained in the adult testes of both species. Staining of these cells became evident around stage IV/V, was prominent in stage VII through IX and declined about stage XII/XIII (rat) or X-XI (mouse). Staining was seen in type A spermatogonia and in elongating spermatids in their cytoplasmatic lobes and along their flagellae. Sertoli cells were unstained. We propose that the pluripotential growth factor bFGF could be involved in the regulation of germ cell proliferation and differentiation in the adult and immature testis.

Hormonal control of pubertal spermatogenesis.

The spermatogenic process of normal rats at 20, 32, and 44 days of age was characterized. Variations in numbers of degenerating and abnormal cells were noted during the cycle in most age groups, indicating a stage-related vulnerability of these cells. The most advanced cell types that were seen at a particular age were frequently abnormal or degenerating. When the numbers of viable cells available to degenerate were considered, the degeneration rate in normal pubertal animals was about 15, 10, and 2 times greater in 20-, 32-, and 44-day-old animals, respectively, than in 75-day-old animals. In 32-day-old rats, neither hypophysectomy nor hypophysectomy and subsequent hormone supplementation resulted in an alteration in the qualitative pattern of germ cell degeneration during the spermatogenic cycle compared with that in the normal animal; however, the treatments did alter the quantitative response of cellular degeneration. Three days posthypophysectomy there was a marked increase in the numbers of total degenerating germ cells. FSH (60 micrograms) given twice daily (as were all hormones) reduced the numbers of degenerating cells significantly, as did LH (13 micrograms). Low dose LH (0.3 micrograms), representing the approximate contaminating dose of LH in the 60-micrograms FSH preparation, and low dose FSH (30 micrograms) did not elicit a response significantly different from that to hypophysectomy alone. LH (13 micrograms) plus FSH (60 micrograms) reduced the levels of degenerating cells such that there was no significant difference from levels in intact 32-day-old rats. The data indicated, for the cell types studied, a lack of specificity of various hormones or hormone combinations in the survival of specific germ cell types. It emphasizes the importance of FSH in pubertal spermatogenesis as well as the synergistic actions of LH and FSH.

Structure/function relationships in active and inactive hamster Leydig cells: a correlative morphometric and endocrine study.

Spermatogenesis can be turned on or off in the seasonally breeding golden (Syrian) hamster in a laboratory setting by exposure of animals to different photoperiod regimens. The present study provides the first detailed quantitative analysis of the subcellular features of hamster Leydig cells during active and inactive phases of spermatogenesis and correlates these features with the endocrine activity of the same animals. Conventional stereological principles and accepted morphometric techniques were used to determine changes in a variety of subcellular constituents of Leydig cells at the extreme phases of gonadal activity produced by maintaining adult hamsters in a long photoperiod (16 h of light, 8 h of darkness) or by exposing them to a short photoperiod (6 h of light, 18 h of darkness) for 12-13 weeks. Compared with Leydig cells from gonadally active animals, Leydig cells obtained from the regressed testis showed a significant reduction in the absolute volume of nearly all of its organelles, including nucleolus (77.0%), mitochondria (50.0%), total endoplasmic reticulum (ER) (86.4%; cisternal ER, 87.0%; tubular ER, 85.5%), lipid (84.2%), peroxisomes (82.5%), multi-vesicular bodies (71.9%), filament-rich area (95.5%), and Golgi complex (69.4%). There was also a significant reduction in organelle surface area, namely outer (76.5%) and inner (72.7%) mitochondrial membrane, total ER (85.0%), cisternal (85.3%) and tubular (84.4%) forms of ER, and Golgi complex (70.0%). The surface areas of the plasma membrane and nuclear membrane were also decreased by 58.3% and 33.7%, respectively. These results demonstrate that the short photoperiod-induced cellular inactivity of Leydig cells is associated with an overall diminution in the volume and surface area of all major organelles, not only those specifically associated with steroid biosynthesis. Virtually every structural parameter of the Leydig cell was significantly and positively correlated with plasma LH levels and with both plasma and testicular concentrations of testosterone. The total content of LH receptors per testis and per Leydig cell was reduced, but the concentration of receptors per unit area of the Leydig cell surface remained unchanged. Correlation of changes in hormonal status with alterations of all Leydig cell organelles suggests a general shut-down of cellular activity in the gonadally regressed animals, rather than a specific effect of pituitary hormones on selected cellular constituents.

Structural manifestations of the rat Sertoli cell to hypophysectomy: a correlative morphometric and endocrine study.

Although the Sertoli cell is a key cell mediating the actions of FSH- and LH-stimulated testosterone (T) in the testis, there is little information to indicate how this cell responds structurally to hormonal insufficiency. The present study used morphometric techniques to study the structural manifestations of the Sertoli cell in adult rats hypophysectomized for 6 and 28 days. Six days posthypophysectomy, a period when germ cell degeneration is first evident, tubular diameter and length, testis weights, volume of the interstitium, and volume of most parameters that comprise the interstitium (except blood vessels) showed significant regressive features. However, virtually no parameter relating to the volume and surface area of the Sertoli cell or its subcellular components was significantly reduced compared with that in the normal animal. Thus, at a time when germ cell degeneration is seen in the testis, the Sertoli cell showed no significant structural response to the changing endocrine status of the animal. In contrast, 28 days after hypophysectomy, virtually all parameters relating to the Sertoli cell and its organelles were significantly decreased compared with those in normal animals. Plasma and tissue testosterone, PRL, and FSH showed a very significant decrease 6 days after hypophysectomy compared with intact animals, but at 28 days there was no further significant decrease in the levels of these hormones. There was no correlation of most organelle volumes and surface areas with endocrine parameters. The size of the Sertoli cell showed positive and significant correlations with the volumes and surface areas of all of its cytoplasmic organelles, except the lipid volume. Short term hypophysectomy resulted in no significant change in either the concentration (femtomoles per mg protein) or the content (femtomoles per testis) of FSH receptors, nor was there a significant change in the number of FSH receptors per cell. However, 28 days after hypophysectomy only the content, not the concentration, of FSH receptors decreased significantly along with a decrease in the number of FSH receptors per cell. Although the marked degenerative changes that are seen in the testis 6 days after hypophysectomy parallel endocrine decline, the Sertoli cell responded slowly from a structural standpoint compared with the Leydig cell. After long term hypophysectomy, significant morphometric changes were observed in all of its structural parameters.

Structural changes in rat Leydig cells posthypophysectomy: a morphometric and endocrine study.

Short and long term responses of the rat Leydig cell were studied posthypophysectomy, at times when germ cell degeneration was first prominent (6 days) and after long term regression of the testis (28 days). In the short term, virtually all structural parameters relating to the volume and surface area of the Leydig cell and its subcellular organelles were significantly lowered compared with those in control animals. Exceptions were the volumes of the nucleolus, heterochromatin, and lysosomes and the surface areas of the nucleus. Structural decreases were generally on the order of 2- to 5-fold in the 6-day period. A statistical analysis of the percent decreases in the short term was performed to determine whether any particular structural features were more sensitive to hypophysectomy than any others. In most instances, no particular organelles were decreased compared to others. However, lipid, although not commonly seen in rat Leydig cells, showed significantly greater percent decreases compared with several other organelles, indicating that the small amount of lipid present is rapidly lost (used) in the short term. After long term hypophysectomy, all structural parameters of the Leydig cell were significantly lowered compared with those in pituitary-intact animals. Only a few parameters (mitochondrial volume, cell surface area, and the surface areas of inner and outer mitochondrial membranes and smooth endoplasmic reticulum) showed more significant decreases in the long term compared with the short term hypophysectomized animals. Most organelle volumes and surface areas correlated positively and significantly with serum and tissue testosterone levels; the exceptions were the volumes of the nucleolus, heterochromatin, lipid, and lysosomes. Compared with the pituitary-intact animal, the content of LH receptors expressed per testis and per Leydig cell was significantly lower in both hypophysectomized groups; however, the number of receptors per given area of individual Leydig cell plasma membrane remained unchanged. Overall, data show that the Leydig cell manifests marked structural changes during early spermatogenic dysfunction.

Murine germ cells do not require functional androgen receptors to complete spermatogenesis following spermatogonial stem cell transplantation.

The spermatogonial stem cell transplantation technique was employed to determine if murine germ cells require functional androgen receptors to complete qualitatively normal spermatogenesis. Testicular cells from testicular feminized mice were injected into the seminiferous tubules of azoospermic mice expressing functional androgen receptors. Recipient testes were analyzed between 110 and 200 days following transplantation. Multiple colonies of complete and qualitatively normal donor-derived spermatogenesis were seen within the seminiferous tubules of each recipient testis, demonstrating that murine germ cells do not require functional androgen receptors to complete spermatogenesis.

Correlative morphology and endocrinology of Sertoli cells in hamster testes in active and inactive states of spermatogenesis.

The seasonally breeding golden (Syrian) hamster, which exhibits photoperiod-dependent transitions between active and inactive states of spermatogenesis, was used as a model to study Sertoli cell structure in the two extreme phases of gonadal activity. The structural parameters of the Sertoli cell and its subcellular organelles were assessed using accepted stereological procedures during active and inactive states of spermatogenesis, and the results correlated with a battery of endocrine parameters obtained from the same animals. Short photoperiod-induced testicular involution was associated with a significant decrease in virtually all morphological parameters of the Sertoli cell, including a dramatic decrease in the volumes and surface areas of the Sertoli cells and their major subcellular organelles. Sertoli cell size and surface area were significantly and positively correlated with the testicular weight, volume of the seminiferous tubule, tubular lumena, tubule diameter, and germ cell numbers. Similar correlations were recorded between the number of germ cells and nearly all subcellular parameters of the Sertoli cell. Only those structural elements that are related to degredative processes (lysosomes and lipid) did not show significant volumetric differences between gonadally active and inactive animals. The observed changes in the structural parameters of the Sertoli cells were significantly correlated with the reduction in plasma levels of FSH, LH, and testosterone and intratesticular levels of testosterone. Exposure of hamsters to a short photoperiod was also associated with an increase in concentration (femtomoles per mg protein), but a decrease in the total content (femtomoles per testis) of testicular FSH receptors. The dissociation of changes in the content and concentration of FSH receptors appears to be related to changes in basal compartment plasma membrane surface areas of the Sertoli cells during testicular regression. The striking changes in Sertoli cell morphology between active and inactive states of spermatogenesis are structural manifestations of alterations in the function of these cells in response to the concomitant endocrine changes in the testis and indicate a virtual shut-down of Sertoli cell function during short photoperiod-induced testicular regression.

Recombinant human follicle-stimulating hormone is capable of exerting a biological effect in the adult hypophysectomized rat by reducing the numbers of degenerating germ cells.

There is considerable controversy as to whether FSH can, under normal circumstances, exert an effect to promote spermatogenesis in the adult rat. Recombinant human FSH (rhFSH) was used to answer a more limited question relating to whether FSH is capable of exerting a biological effect in promoting adult spermatogenesis. Can a pure preparation of FSH prevent the regressive changes seen after hypophysectomy (Hx) in a short term experiment? To answer this question, five groups of adult rats were used as follows: pituitary-intact animals, 3-day hypophysectomized (Hx), 3-day Hx given 3 mg testosterone propionate (T)/day for 7 days, 3-day Hx given 22 IU rhFSH for 7 days, and 3-day Hx given saline vehicle for 7 days. Testis weight, seminiferous tubule diameter, analysis of four degenerating germ cell types, the relative amount of lipid, and the levels of FSH receptors showed that FSH could, in a significant manner, prevent the regressive changes accompanying Hx. FSH was not as effective as T in doing so, because the FSH values were always intermediate between T-maintained animals and those after long term Hx. The Leydig cell was eliminated as a possible source of FSH-stimulated T promotion of spermatogenesis, given that morphometry and tissue T assays indicated that no additional production of T was elicited by rhFSH. The assay system used to enumerate degenerating germ cells proved a very sensitive indicator of the ability of hormones to maintain cell viability in short term experiments. The data not only show that FSH can exert a biological effect, but that this effect is qualitatively similar to that seen after the administration of T in terms of the maintenance of viability of specific germ cell types. A hypothesis is presented whereby FSH and T, although the former acting by a second messenger system and the latter by binding to nuclear receptors, can stimulate the genome to exert similar qualitative effects promoting the viability of germ cells.

Enhancement of A spermatogonial proliferation and differentiation in irradiated rats by gonadotropin-releasing hormone antagonist administration.

The initial changes in the numbers, proliferation, and differentiation of A spermatogonia in irradiated rats after the administration of a GnRH antagonist, which is known to induce differentiation in this system, were investigated. LBNF1 rats were given 6 Gy of gamma-irradiation; some were treated with the GnRH antagonist Cetrorelix beginning 15 weeks after irradiation. Although the spermatogonia in the irradiated rats without hormone treatment continue to proliferate (labeling and mitotic indexes of 24% and 18%, respectively), they underwent apoptosis (apoptotic indexes of 21% by the terminal transferase-mediated end labeling assay and 9% by nuclear morphology), resulting in a constant number of A spermatogonia. Whole mount analysis of clones ofA spermatogonia revealed that larger clones were more likely to undergo apoptosis than mitosis. Hormone administration decreased the intratesticular testosterone concentration to 6% of the level in irradiated rats within 1 week. Concomitantly, there was a decrease in spermatogonial apoptotic indexes to 43% of levels in irradiated-only rats, leading to an increases in their numbers by 150%, their diameters by 11%, and their labeling indexes by 31%. The sizes of the mitotic clones gradually increased, and clones of more than eight cells appeared at week 3 of hormone treatment. A spermatogonial differentiation began at week 4, and by week 6.6, differentiation occurred in 30% of the tubules. Thus, suppression of intratesticular testosterone by the GnRH antagonist may be responsible for the immediate changes in spermatogonial numbers and kinetics, but several additional steps are required before differentiation begins, which did not occur until week 4.

Blockage of the rete testis and efferent ductules by ectopic Sertoli and Leydig cells causes infertility in Dax1-deficient male mice.

DAX-1, an X-linked member of the orphan nuclear receptor superfamily of transcription factors, plays a key role in sex determination and gonadal differentiation. Dax1-deficient male mice are infertile and have small testes despite normal serum levels of T and gonadotropins. Examination of Dax1-deficient testes reveals dilated seminiferous tubules and abnormal parameters of sperm fertilizing capability consistent with a possible obstruction in the testis. To test this hypothesis, we performed a comprehensive evaluation of the male reproductive tract in Dax1-deficient mice. Light and electron microscopic examination revealed the rete testis is blocked by aberrantly located Sertoli cells, creating a tailback of necrosing sperm in the testis. Sertoli cells also obstruct the proximal and middle efferent ductules, and this is accompanied by an overgrowth of the efferent duct epithelium. Seminiferous tubules close to the rete testis contain ectopic Leydig cells, distinct from the hyperplastic Leydig cells present in the interstitial space. The peritubular tissue surrounding these tubules is frequently abnormal, containing relatively undifferentiated myoid cells and no basement membrane between the myoid cells and Sertoli cells. A third of aged (>1-yr-old) Dax1-deficient male mice develop sex cord-stromal tumors, derived from cells of the Sertoli/granulosa cell or Leydig cell lineages. Combined, these observations reveal abnormal differentiation and proliferation of Leydig cells and Sertoli cells in Dax1-deficient male mice, leading to obstruction of the rete testis and infertility.

Testicular endocrine function in GH receptor gene disrupted mice.

The consequences of disruption of GH receptor gene in GH receptor knockout mice on testicular function were evaluated. Adult male GH receptor knockout mice and their normal siblings were divided in to two subgroups and treated with either saline or ovine LH (0.3 microg/g BW) in saline. One hour after saline or LH administration, blood was obtained via heart puncture. Plasma IGF-I, LH, FSH, PRL, androstenedione, and testosterone levels were measured by RIAs. Testicular LH and PRL receptor numbers as well as pituitary LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were measured. Also, testicular morphometric analysis was performed. Unlike in normal, wild-type mice, the circulating IGF-I was undetectable in GH receptor knockout mice. The plasma PRL levels were (P<0.01) higher in GH receptor knockout mice than in their normal siblings. The basal LH secretion was similar in normal and GH receptor knockout mice. However, the circulating FSH levels were lower (P<0.001) in GH receptor gene disrupted mice. Administration of LH resulted in a significant (P<0.001) increase in plasma testosterone levels in both GH receptor knockout and normal mice. However, this testosterone response was attenuated (P < 0.01) in GH receptor knockout mice. Plasma androstenedione responses were similar in both GH receptor knockout and normal mice. Testicular LH and PRL receptor numbers were significantly decreased in GH receptor knockout mice. The results of the morphometric analysis of the testis revealed that the Leydig cell volume per testis was reduced in mice with GH receptor gene disruption. The steady-state of LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were not different in GH receptor knockout mice relative to their normal siblings. The present in vivo study demonstrates that in GH receptor knockout mice, LH action on the testis in terms of testosterone secretion is significantly attenuated and suggests that this is due to a decrease in the number of testicular LH receptors. The reduced number of PRL receptors may contribute to the diminished responsiveness of testicular steroidogenesis to LH by decreased ability to convert androstenedione to testosterone. These changes are most likely due to the absence of circulating IGF-I. These findings provide evidence that systemic IGF-I plays a major modulatory role in testicular endocrine function.

Visualization of anti-sperm plasma membrane IgG and Fab as a method for localization of boar sperm membrane antigens.

A simple method for ultrastructural localization of sperm surface antigens by direct visualization of bound antibodies is presented. Anti-sperm plasma membrane (ASPM) immunoglobulin (Ig) G, visualized in tissues treated with an osmium:ferrocyanide mixture, projected 11-13 nm from the surface and ASPM Fab fragments projected 8-10 nm from the surface. The density of IgG labeling, as subjectively estimated, corresponded to indirect immune fluorescein isothiocyanate, indirect immunoferritin, and sperm-vesicle labeling patterns. Agglutination of sperm vesicles and sperm were demonstrated and the linking antibody visualized. A second antibody on protein A directed against ASPM IgG made the immunologic tag more apparent and indicated, in disrupted sperm preparations, labeling of both sides of the plasma membrane. The method provides for easy and sensitive localization of sperm surface antigens at the ultrastructural level and is presently being used to localize specific sperm antigens.

Spermatogonial transplantation - an update for the millennium.

Spermatogonial transplantation as developed in the laboratory of Ralph Brinster has been a technological breakthrough in the study of Sertoli-germ cell interactions. For the first time, germ cells can be transferred from one animal to another and from one species to another. The transfer technology combined with developments in freezing germ cells, long-term culture of germ cells, and enrichment of stem cell populations portend even more significant breakthroughs in the new millennium. The ultimate application of germ cell transfer would allow the in vitro genetic manipulation of cultured stem cells that could then be transplanted into recipient syngeneic or xenogeneic recipients and give rise to functional male gametes. Clearly, this achievement would have applications in basic science, human medicine, and domestic and wild animal reproduction. While progress in this direction has been significant and swift, significant barriers such as immunological response and mechanisms for introducing genetic material into the stem cells remain to be examined. This report is a chronological review of the technological advances made and conceptual insights gained since the first report of successful transplantation in 1994.

Radiation-induced cell death in the mouse testis: relationship to apoptosis.

The killing of male germ cells by radiation and other toxicants has recently been attributed to apoptosis, but a critical evaluation of the presence of the different features of apoptosis has not been performed. In this study, mouse testes exposed to radiation were examined by light microscopy, electron microscopy and terminal transferase-mediated end labeling (TUNEL) to determine whether the cells were apoptotic according to several criteria. Testes were irradiated with single doses of gamma rays of up to 5 Gy. Although the maximum response was produced by 5 Gy, even 0.5 Gy induced marked changes. The numbers of abnormal spermatogonia reached a peak 12 h after irradiation and then declined, and the total number of spermatogonia began to decline at 12 h. These changes were most prominent among the B spermatogonia and early preleptotene spermatocytes. When examined by both light and electron microscopy, the majority of the abnormal spermatogonia showed condensation of nuclear chromatin and some showed features similar to necrosis, but the typical morphological characteristics of apoptosis, margination of chromatin and nuclear fragmentation, were rare. Many of the abnormal spermatogonia were TUNEL-positive, with the maximum number occurring at 12 h after irradiation. Although the morphological features of radiation-induced spermatogonial degeneration were not typical of apoptosis, the TUNEL staining, the rapid onset of degeneration and the sensitivity to low doses suggest that the mechanism of radiation-induced spermatogonial degeneration is closely related to apoptosis.

Improvement in the quality and fertilization potential of a human sperm population using the rise technique.

Semen from 63 individuals participating in an in vitro fertilization program was processed using a modified rise technique. Overall normal morphology was significantly improved in the rise (79.2%) as compared with the unprocessed sample (57.8%), and six of seven specific morphologic abnormalities were significantly reduced. Motility was significantly enhanced from 51.8% in the unprocessed samples to 89.1% in the rise samples. Spermatozoa recovered in the rise portion of the sample represented 5.9% of the total sample. Ultrastructural morphometry revealed that the rise was relatively free of abnormal sperm forms, acellular debris and non-sperm cellular elements as compared with the non-rise portion of the sample or a typical unprocessed sample. Volume density measurements demonstrated that only 18.1% of the volume of the non-rise sample was composed of normal spermatozoa compared with 83.4% of the volume of the rise. In a separate set of experiments utilizing 21 samples, the penetration of hamster eggs was significantly enhanced from 37.9% to 67.2% using spermatozoa from the initial washed sample and those from the rise, respectively. These data demonstrate the qualitative and quantitative improvements, as well as the increase in fertilizing potential, of the rise portion of the semen sample.

Ultrastructural observations of spermatogenesis following transplantation of rat testis cells into mouse seminiferous tubules.

The testes of busulfan-treated immunodeficient mice receiving seminiferous tubule injections of testis cells from rats were examined by light and electron microscopy. The presence of active rat spermatogenesis was verified by criteria that are known to characterize spermatogenic cells of this species. In addition, spermatogenesis from the mouse was identified as taking place in some seminiferous tubules as the result of reinitiation of spermatogenesis after busulfan treatment. Rat spermatogenesis in mouse seminiferous tubules showed the generally recognized associations of cells known to characterize stages of spermatogenesis of the rat. The Sertoli cells associated with rat spermatogenesis were identified ultrastructurally as being of mouse origin. Thus, rat spermatogenesis, which has a cycle length that is 50% longer than mouse spermatogenesis, can proceed among mouse Sertoli cells, which supposedly exert much shorter cyclic influences in concert with mouse germ cell development. Studies are needed to determine if the timing of rat spermatogenesis is controlled by the germ cells or the Sertoli cells. These observations are considered preliminary since a thorough study of somatic-germ cell relationships was not undertaken. It is concluded that a mouse Sertoli cell in the environment provided by the mouse testis can produce both mouse and rat gametes.