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Ectoplasmic specializations are actin filament-endoplasmic reticulum complexes that occur in Sertoli cells at sites of intercellular attachment. At sites between inter-Sertoli cell attachments, near the base of the cells, the sites are also related to tight junctions. We studied the characteristics of ectoplasmic specializations from six species using conventional views in which thin sections were perpendicular to the plane of the membranes, we used rare views in which the sections were in the plane of the membrane (en face views), and we also used the freeze-fracture technique. Tissues postfixed by osmium ferrocyanide showed junctional strands (fusion points between membranes) and actin bundles, actin sheets, or both, which could be visualized simultaneously. En face views demonstrated that the majority of tight junctional strands ran parallel to actin filament bundles. Usually, two tight junctional strands were associated with each actin filament bundle. Parallel tight junctions were occasionally extremely close together ( approximately 12 nm apart). Tight junctional strands were sometimes present without an apparent association with organized actin bundles or they were tangential to actin bundles. En face views showed that gap junctions were commonly observed intercalated with tight junction strands. The results taken together suggest a relationship of organized actin with tight junction complexes. However, the occasional examples of tight junction complexes being not perfectly aligned with actin filament bundles suggest that a precise and rigidly organized actin-tight junction relationship described above is not absolutely mandatory for the presence or maintenance of tight junctions. Species variations in tight junction organization are also presented.

ROSA22 male mice are sterile due to a recessive gene-trap mutation that affects development of the spermatid flagellum. The defect involves the flagellar axoneme, which becomes unstable around the time of its assembly. Despite a subsequent complete failure in flagellar assembly, development of the spermatid head appears normal and the spermatid head is released at the correct stage in spermatogenesis. The mutation is pleiotropic. Although ROSA22 homozygote males have normal levels of circulating testosterone and display normal mating behavior, they do not exhibit intermale aggressive behavior and have reduced body fat. The mutated gene (Gtrgeo22) maps to mouse chromosome 10 and is closely flanked by two known genes, Madcam1 and Cdc34. Ribonuclease protection analysis indicates that expression of the flanking genes is unaffected by the mutation. Gtrgeo22 is expressed at low levels in epithelial cells in several tissues, as well as in testis and brain. Analysis of the peptide coding sequence suggests that Gtrgeo22 encodes a novel transmembrane protein, which contains dileucine and tyrosine-based motifs involved in intracellular sorting of transmembrane proteins. Analysis of the Gtrgeo22 gene product should provide novel insight into the molecular basis for intermale aggression and sperm flagellar development.

Spermatogenesis is a complex process in which spermatogonial stem cells divide and subsequently differentiate into spermatozoa. This process requires spermatogonial stem cells to self-renew and provide a continual population of cells for differentiation. Studies on spermatogonial stem cells have been limited due to a lack of unique markers and an inability to detect the presence of these cells. The technique of germ cell transplantation provides a functional assay to identify spermatogonial stem cells in a cell population. We hypothesized that vitamin A-deficient (VAD) and hyperthermically treated testes would provide an enriched in vivo source of spermatogonial stem cells. The first model, hyperthermic treatment, depends on the sensitivity of maturing germ cells to high temperatures. Testes of adult mice were exposed to 43 degrees C for 15 min to eliminate the majority of differentiating germ cells. Treated donor testes were 50% of normal adult testis size and, when transplanted into recipients, resulted in a 5.3- and 19-fold (colonies and area, respectively) increase in colonization efficiency compared to controls. The second model, VAD animals, also lacked differentiating germ cells, and testes weights were 25% of control values. Colonization efficiency of germ cells from VAD testes resulted in a 2.5- and 6.2-fold (colonies and area, respectively) increase in colonization compared to controls. Hyperthermically treated mice represent an enriched source of spermatogonial stem cells. In contrast, the low extent of colonization with germ cells from VAD animals raises important questions regarding the competency of stem cells from this model.

Bax is a multidomain, proapoptotic member of the Bcl-2 family that is required for normal spermatogenesis in mice. Despite its proapoptotic function, previous results found that Bax-deficient mature male mice demonstrate increased cell death and dramatic testicular atrophy. The present study examined the role of Bax during the normal development of the testis to determine whether the increased cell death in mature mice could be explained by decreased apoptosis earlier in development. Consistent with this hypothesis, testicular atrophy is preceded by increased testicular weight and hypercellular tubules in immature Bax-deficient mice. TUNEL staining at Postnatal Day (P) 7 and morphological quantitation between P5 and P15 demonstrates decreased germ cell apoptosis in Bax-deficient mice. By P15, increased numbers of type A spermatogonia, and at P12 and P15, an increase in intermediate type spermatogonia were noted in Bax-deficient animals. By P25, the number of basal compartment cells was greatly increased in Bax-deficient animals compared with controls such that four or five layers of preleptotene spermatocytes were routinely present within the basal compartment of the testis. Although the Sertoli cell barrier was significantly removed from the basement membrane, it appeared intact as judged by the hypertonic fixation test. During late pubertal development, massive degeneration of germ cells took place, including many of those cell types that previously survived in the first wave of spermatogenesis. The data indicate that Bax is required for normal developmental germ cell death in the type A spermatogonia, specifically dividing (A(2), A(3), and A(4)) spermatogonia, at a time at which the number of spermatogonia is regulated in a density-dependent manner. The massive hyperplasia that occurs in Bax-deficient mice subsequently results in Bax independent cell death that may be triggered by overcrowding of the seminiferous epithelium.

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.

Desert hedgehog (Dhh) is a cell-signaling molecule that was first discovered in Drosophila. A unique testicular phenotype has been described in neonatal and adult Dhh-null animals that includes anastomotic seminiferous tubules, pertitubular cell abnormalities, and absence of adult-type Leydig cells. In the present study, we addressed the developmental basis for the abnormalities previously described for the adult Dhh-null phenotype. The source of Dhh is the Sertoli cell, and receptors are localized on peritubular cells and possibly Leydig cells. The development of testes from Dhh-null mouse embryos was studied using light and electron microscopy at 11.5, 12.5, 13.5, and 16.5 days postcoitum (dpc) and was compared with that in control Dhh heterozygous and wild-type embryos. Dhh-null and control testes were generally similar during the period of early cord formation (11.5-12.5 dpc). By 13.5 dpc, the basal lamina delimiting the cords was lacking in some regions and disorganized in Dhh-null testes, and occasional germ cells were seen outside cords. At 16.5 dpc, these defects were more prominent and cord organization was less well defined than in controls. In addition, there were numerous extracordal germ cells, some of which were partially enclosed by a somatic cell of unknown identity. Numerous fibroblast-like cells, apparently secreting collagen and basal lamina, characterized the interstitium of the Dhh-null testis. These defects likely stem from abnormal peritubular stimulation due to the lack of Dhh, leading to the abnormalities seen in the developmental stages studied here and in the adult testis.

Characteristics of spermatogonia were determined in the C57BL/6J strain mouse using high-resolution light microscopy of plastic-embedded tissues and identifying cells during stages of the spermatogenic cycle. The frequency of expecting each spermatogonial cell type was a major factor in identifying and categorizing various cell types. Although numerous characteristics were described, several major differences were noted in spermatogonial cell types. The group comprising A(s), A(pr), and A(al) spermatogonia could be differentiated based primarily on mottling of heterochromatin throughout the nucleus in the absence of heterochromatin lining the nuclear envelope. The A(1) cells displayed finely granular chromatin throughout the nucleus and virtually no flakes of heterochromatin along the nuclear membrane. The A(2) through A(4) spermatogonia contained progressively more heterochromatin rimming the nucleus. Intermediate-type spermatogonia displayed flaky or shallow heterochromatin that completely rimmed the nucleus. Type B spermatogonia showed rounded heterochromatin periodically along the nuclear envelope. Use of gray-scale histograms allowed objective quantification of nuclear characteristics and showed a logical shift in the gray scale to a narrower and darker profile, from four cell types leading to A(1) cells. The ability to differentiate spermatogonial types is a prerequisite to studying the behavior and kinetics of the earliest of the germ cell types in both normal and abnormal spermatogenesis.

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.

The distribution of type A spermatogonia was studied using drawings of cross-sectioned tubules at various stages of the spermatogenic cycle of perfusion-fixed, epoxy-embedded mouse testis. Spermatogonia were classified as either positioned opposite the interstitium or opposite the region where two tubules make contact or in a defined, intermediate region at which the two tubules diverged. At stage V, the population of type A spermatogonia, comprised of A(s) through A(al) cells, is randomly positioned around the periphery of the seminiferous tubule. The A(s) through A(al) population becomes nonrandomly distributed beginning at stage VI, being located primarily in regions where the tubule opposes the interstitium, and remains nonrandom through stage III of the next cycle. The A(1) spermatogonia of stage VII, derived from most A(pr) and A(al) spermatogonia, and the A(2) spermatogonia of stage IX, derived from the A(1) spermatogonia, are also nonrandomly positioned opposing the interstitium. However, the A(3) population of stage XI becomes randomly distributed around the tubule. To our knowledge, these are the first data to show that the more primitive spermatogonial types (A(s) to A(al)) move to specific sites within the seminiferous tubule. Division of the regularly spaced, more primitive spermatogonia (A(s) to A(al)) leads to the spread of their progeny (A(1) to A(4)) laterally along the base of the seminiferous tubule. The lateral spread from more or less evenly spaced foci ensures that spermatogenesis is conducted uniformly around the entire tubule. The data also suggest that the position of a seminiferous tubule in the mouse is stabilized in relationship to other seminiferous tubules.

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.

Bclw is a death-protecting member of the Bcl2 family of apoptosis-regulating proteins. Mice that are mutant for Bclw display progressive and nearly complete testicular degeneration. We performed a morphometric evaluation of testicular histopathology in Bclw-deficient male mice between 9 days postnatal (p9) through 1 yr of age. Germ cell loss began by p22, with only few germ cells remaining beyond 7 mo of age. A complete block to elongated spermatid development at step 13 occurred during the first wave of spermatogenesis, whereas other types of germ cells were lost sporadically. Depletion of Sertoli cells commenced between p20 and p23 and continued until 1 yr of age, when few, if any, Sertoli cells remained. Mitochondria appeared to be swollen and the cytoplasm dense by electron microscopy, but degenerating Bclw-deficient Sertoli cells failed to display classical features of apoptosis, such as chromatin condensation and nuclear fragmentation. Macrophages entered seminiferous tubules and formed foreign-body giant cells that engulfed and phagocytosed the degenerated Sertoli cells. Leydig cell hyperplasia was evident between 3 and 5 mo of age. However, beginning at 7 mo of age, Leydig cells underwent apoptosis, with dead cells being phagocytosed by macrophages. The aforementioned cell losses culminated in a testis-containing vasculature, intertubular phagocytic cells, and peritubular cell "ghosts." An RNA in situ hybridization study indicates that Bclw is expressed in Sertoli cells in the adult mouse testis. Consequently, the diploid germ cell death may be an indirect effect of defective Sertoli cell function. Western analysis was used to confirm that Bclw is not expressed in spermatids; thus, loss of this cell type most likely results from defective Sertoli cell function. Because Bclw does not appear to be expressed in Leydig cells, loss of Leydig cells in Bclw-deficient mice may result from depletion of Sertoli cells. Bclw-deficient mice serve as a unique model to study homeostasis of cell populations in the testis.

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.

Testes from adult and prepubertal mice lacking the Desert hedgehog (DHH:) gene were examined in order to describe further the role of Dhh in spermatogenesis because, in a previous report, DHH:-null male mice were shown to be sterile. Dhh is a signaling molecule expressed by Sertoli cells. Its receptor, patched (Ptc), has been previously localized to Leydig cells and is herein described as being localized also to peritubular cells. Two phenotypes of the mice were observed: masculinized (7.5% of DHH:-null males) and feminized (92.5%), both of which displayed abnormal peritubular tissue and severely restricted spermatogenesis. Testes from adult feminized animals lacked adult-type Leydig cells and displayed numerous undifferentiated fibroblastic cells in the interstitium that produced abundant collagen. The basal lamina, normally present between the myoid cells and Sertoli cells, was focally absent. We speculate that the abnormal basal lamina contributed to other characteristics, such as extracordal gonocytes, apolar Sertoli cells, and anastomotic seminiferous tubules. The two DHH:-null phenotypes described have common peritubular cell defects that may be indicative of the essential role of peritubular cells in development of tubular morphology, the differentiation of Leydig cells, and the ultimate support of spermatogenesis.

Cimetidine (Tagamet) is a potent histaminic H2-receptor antagonist, extensively prescribed for ulcers and now available without prescription. Cimetidine is a known testicular toxicant, but its mechanism of action remains uncertain. Rats were treated i.p. with cimetidine either at 50 mg/kg or 250 mg/kg body weight for 59 days. Accessory sex organ weights, but not testis weight, were significantly reduced in the high dose treated groups. FSH levels were significantly elevated in both treated groups, but testosterone levels were unchanged. A high degree of variability characterized testis histology, with most tubules appearing normal and some tubules (15-17%) partially lacking or devoid of germ cells. Morphometry showed that although seminiferous tubule volume was not significantly changed, the volume of peritubular tissue was reduced in the high dose group. There was extensive duplication of the basal lamina, lamina densa in both apparently normal spermatogenic tubules and severely damaged tubules. Apoptotic peritubular myoid cells were also found. TUNEL labeling confirmed extensive apoptotic cell death in peritubular cells, but revealed apoptosis of vascular smooth muscle. Given that 1) peritubular myoid cell apoptosis occurs in apparently normal tubules, that 2) basal lamina disorders are found, and that 3) peritubular cells are lost from the testis, it is suggested that the primary event in cimetidine-related damage is targeted to testicular smooth muscle cells. This is the first in vivo-administered toxicant to be described that targets myoid cells, resulting in abnormal spermatogenesis.

In mice, the juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis followed by failure of type A spermatogonial stem cells to repopulate the testis, rendering male animals sterile. It is not clear whether the defect in jsd resides in a failure of the somatic component to support spermatogenesis or in a failure that is intrinsic to the mutant's germ cells. To determine if the jsd intratesticular environment is capable of supporting spermatogenesis, germ cell transplantation experiments were performed in which C57BL/6 ROSA germ cells were transplanted into jsd recipients. To determine if jsd spermatogonia are able to develop in a permissive seminiferous environment, jsd germ cells were transplanted into W/W(v) and busulfan-treated C57BL/6 animals. The data demonstrate that up to 7 mo after transplantation of normal germ cells, jsd seminiferous tubules are capable of supporting spermatogenesis. In contrast, when jsd germ cells were transplanted into busulfan-treated C57BL/6 testis, or into testis of W/W(v) mice, no jsd-derived spermatogenesis was observed. The data support the hypothesis that the jsd phenotype is due to a defect in the germ cells themselves, and not in the intratubular environment.

Spermatogonial stem cell transplantation was first reported by Ralph Brinster's laboratory in 1994. It has proven to be a technological breakthrough in the study of both stem cells and Sertoli cell-germ cell interactions. This technique can be used to transfer testicular stem cells successfully from one animal to another of the same species (referred to as syngeneic transplants) and sometimes to an animal of a different species (xenogeneic transplants). This transfer technique, combined with developments in cryopreservation, long-term culture, and the enrichment of stem cell populations makes more significant breakthroughs likely in the near future. Ultimately, the application of spermatogonial stem cell transfer will allow transplantation of cultured stem cells manipulated genetically in vitro to give rise to functional male gametes with an altered genotype. This achievement will have applications in basic science, human medicine, and domestic and wild animal reproduction. Although progress toward this goal has been swift, potentially significant barriers, such as the stable incorporation of genetic material into stem cells and immunological responses to the introduced germ cells, remain to be overcome. This article is a review of the scientific advances made since the initial report of successful transplantation in 1994.

Cell type-specific microtubules, such as the Sertoli cell microtubules and the manchette and flagellum microtubules of the spermatids, play essential roles in spermatogenesis. We identified the gene encoding E-MAP-115 (epithelial microtubule-associated protein of 115 kD) as a retinoic acid-inducible gene using gene trap mutagenesis in mouse embryonic stem cells. The gene trap insertion led to a null allele of the E-MAP-115 gene and, in agreement with its high expression in the testis, male mice homozygous for the mutation were sterile because of deformation of spermatid nuclei and subsequent gradual loss of germ cells. Consistent with a possible role for E-MAP-115 in stabilizing microtubules, microtubule associations in the mutant were morphologically abnormal in the manchette of spermatids and in Sertoli cells. We hypothesize that the abnormal microtubules in these two cell types are responsible for deformation of spermatid nuclei and germ cell loss, respectively, and indicate an essential role for E-MAP-115 in microtubule functions required for spermatogenesis.

Transition nuclear proteins (TPs), the major proteins found in chromatin of condensing spermatids, are believed to be important for histone displacement and chromatin condensation during mammalian spermatogenesis. We generated mice lacking the major TP, TP1, by targeted deletion of the Tnp1 gene in mouse embryonic stem cells. Surprisingly, testis weights and sperm production were normal in the mutant mice, and only subtle abnormalities were observed in sperm morphology. Electron microscopy revealed large rod-like structures in the chromatin of mutant step 13 spermatids, in contrast to the fine chromatin fibrils observed in wild type. Steps 12-13 spermatid nuclei from the testis of Tnp1-null mice contained, in place of TP1, elevated levels of TP2 and some protamine 2 (P2) precursor. Most of the precursor was processed to mature P2, but high levels of incompletely processed forms remained in epididymal spermatozoa. Sperm motility was reduced severely, and approximately 60% of Tnp1-null males were infertile. We concluded that TP1 is not essential for histone displacement or chromatin condensation. The absence of TP1 may partially be compensated for by TP2 and P2 precursor, but this dysregulation of nucleoprotein replacement results in an abnormal pattern of chromatin condensation and in reduced fertility.

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.

Development of spermatogonial transplants was studied by using 5- to 6-wk-old histocompatible mice as cell donors and sterile (W-locus) mice as recipients. Groups of animals transplanted with germ cell suspensions were killed at 10 min, 9 h, 24 h, 1 wk, 1 mo, 2 mo, and 3 mo along with age-matched "start" and "end" W-locus controls. Weight of testes increased significantly at 24 h through 3 mo after germ cell transplantation, suggesting that the infused cells quickly stimulated organ function. Small clones of young spermatocytes were evident at 1 mo and sperm at 2 mo. The percentage of tubular profiles containing active spermatogenesis originating from spermatogonia increased with time (0.8% at 1 mo, 8.9% at 2 mo, and 28.2% at 3 mo). Most transplanted germ cells were eliminated from the seminiferous epithelium through phagocytosis by Sertoli cells that occurred primarily before 1 wk, although some pachytene cells were able to proceed through meiosis by 1 wk. A variety of abnormal features are described that characterize developing spermatogenesis in the transplanted testis. Spermatogenesis improved quantitatively and qualitatively with time although released sperm were frequently engulfed by intratubular macrophages and Sertoli cells. A quantitative analysis of spermatogenesis from transplanted germ cells will serve as a basis for improving spermatogonial transplant efficiency.

Spermatogenesis is one of the most productive self-renewing systems in the body: on the order of 10(7) spermatozoa are produced daily per gram of testis tissue. In each mammalian species, the time required for completion of the process is unique and unalterable. Because the process is supported by somatic Sertoli cells, it has generally been thought that cell-cell interaction between germ and Sertoli cells controls the duration of cell cycles and cellular organization. We have used the newly developed technique of spermatogonial transplantation to examine which cell type(s) determines the rate at which germ cells proceed through spermatogenesis. Rat germ cells were transplanted into a mouse testis, and the mouse was killed 12.9-13 days after administration of a single dose of [3H]thymidine. The most advanced rat cell type labeled was the pachytene spermatocyte at stages VI-VIII of the spermatogenic cycle. In animals given only rat cells, some endogenous spermatogenesis of the mouse recovered. The most advanced labeled mouse cell types in recipients killed 12.9-13 days after administration of a single dose of [3H]thymidine were meiotic cells or young spermatids, which is consistent with a spermatogenic cycle length comparable to the 8.6 days reported for the mouse. The same results were obtained if a mixture of rat and mouse cells were transplanted. There existed two separate timing regimens for germ cell development in the recipient mouse testis; one of rat and one of mouse duration. Rat germ cells that were supported by mouse Sertoli cells always differentiated with cell cycle timing characteristic of the rat and generated the spermatogenic structural pattern of the rat, demonstrating that the cell differentiation process of spermatogenesis is regulated by germ cells alone.

Spermatogenesis continues after long-term hypophysectomy (Hx), but massive cell degeneration prevents seminiferous tubules from attaining the full complement of cells. One objective of this study was to determine the vulnerable sites for completion of spermatogenesis in long-term Hx rats. It is now known that Leydig cells continue to secrete small amounts of androgen after Hx. A second objective was to determine the cellular sites that are maintained by residual androgen secreted by Leydig cells post-Hx. Two groups of adult animals were utilized. Both groups were Hx for 36 days, but one group of rats received the androgen antagonist flutamide during the 26th through the 36th day of Hx (10 days). Germ-cell numbers were quantified using a method that allowed their expression as numbers of cells present per hour of development. In the long-term Hx rat, the germ-cell population increased to preleptotene, but the divisions that led to preleptotene were inefficient due to cell degeneration. Subsequent to preleptotene, there was a gradual loss in cells such that there were few germ cells remaining by steps 9-13. Flutamide given to Hx rats did not result in a significant difference in the numbers of intermediate and type B spermatogonia or significant differences in progenitor cells. A significant and major depression of cell numbers in Hx-flutamide-treated rats occurred in the cell division of type B spermatogonia to form preleptotene spermatocytes. There was a less dramatic, although significant, depression of cell numbers in Hx-flutamide-treated animals that occurred from preleptotene until late pachytene as well as an increased loss of round spermatids at midcycle (step 5-6). These data demonstrate that cell loss after long-term Hx occurs at numerous phases of spermatogenesis. The data also demonstrate that the presence of residual androgen action after long-term Hx results in enhanced germ-cell survival. Although the major blockage in cell viability occurs at midcycle steps in the long-term Hx rat, there are several other hormone-sensitive phases of spermatogenesis.

A quantitative analysis of germ-cell populations in normal, hypophysectomized (Hx), and Hx-hormone-treated animals was undertaken during periods of regression that were characterized as intermediate, between short-term and long-term regression of the testis. Twenty-one groups of adult rats were administered either follicle-stimulating hormone (FSH), growth hormone, thyroid-stimulating hormone (TSH), or testosterone (T) in various doses and combinations. The dosage of T administered was less than that expected to achieve maximum testis weight. Flutamide and Casodex were used to compete with androgen binding to receptors in Hx animals, as it is known that small amounts of androgen are secreted in the absence of pituitary stimulation. Follicle-stimulating hormone, T, and TSH all significantly maintained testis weight as compared with Hx controls, although FSH and T, singly or in combination, were the most effective. Contamination of the TSH preparation with trace amounts of FSH was apparently responsible for the slight maintenance of testis weight. A novel assay for determination of the numbers of viable germ cells was used in a subset of these groups to determine the cellular sites of FSH and T action. Numbers of type A spermatogonia were lowered after Hx and were maintained by either FSH or T or a combination of these hormones. Other phases of germ-cell development most susceptible to FSH and/or T were the successive conversions of type A spermatogonia to intermediate spermatogonia, intermediate spermatogonia to type B spermatogonia, preleptotene spermatocytes to pachytene spermatocytes, and early pachytene spermatocytes to intermediate maturity pachytene spermatocytes during early and midcycle phases of pachytene spermatocyte development. Germ-cell loss during meiosis and virtually every phase of spermatid development was largely prevented by FSH or T or a combination of these hormones. Thus, in testes in advanced stages of regression, both FSH and T were capable of preventing cell loss, suggesting that both hormones can affect the survival of the same cell type. The present study demonstrated that FSH can partially compensate for lowered T levels. The combined administration of FSH and T was more effective in preventing overall cell degeneration than either hormone alone. Unlike the initial phase of spermatogenesis, in which there is a largely midcycle loss of germ cells due to Hx, the loss of cells during testis regression is more widespread and impacts several cell types in more than one stage of the spermatogenic cycle.

The potential for follicle-stimulating hormone (FSH) to promote germ-cell survival and the cellular sites of FSH action were studied using a gonadally maturing (pubertal), hypophysectomized (Hx) rat model in which residual testosterone (T) activity was blocked by injections of an androgen-receptor antagonist, flutamide. Recombinant human FSH was given to androgen-deprived and androgen-blocked male rats at 27 days of age to determine maintenance of individual germ-cell types at 35 days of age. Follicle-stimulating hormone significantly increased testis weights and tubular diameters as compared with Hx and Hx-flutamide controls, although testis weights in FSH-treated animals were significantly lower than in pituitary-intact animals. Morphometric assays to determine ratios of germ cells to Sertoli cells and to determine the number of germ cells present per hour of development showed that the population of type A spermatogonia in the early stages of the cycle was not responsive to FSH. Follicle-stimulating hormone had a marked ability to maintain cell viability in the rapid, successive divisions that begin in the latter part of the cycle and that continue through the next cycle (i.e., from type A1 to A4 and from intermediate spermatogonia to type B spermatogonia to preleptotene spermatocytes to leptotene/zygotene spermatocytes to young pachytene spermatocytes). The data also suggest T responsiveness of these cell types since the Hx-FSH-flutamide group showed lower cell viability at the aforementioned steps when compared with the Hx-FSH group. Too few cell types were present at subsequent phases of spermatogenesis to allow a sensitive determination of FSH activity in the maintenance of cell viability. The data show the potential of FSH in the absence or relative absence of T activity to maintain cell viability. These data support the concept of overlapping and synergistic (or additive) effects of T and FSH in the immature rat and identify the cellular sites of FSH action.

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.

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.

The objective of the present study was to provide a morphological characterization of spermatogenesis following germ cell transplantation into the seminiferous tubular lumen of another mouse. The recipient mice (W-locus) were sterile because of a defect in spermatogenesis resulting from the failure of virtually all germ cell precursors to migrate to the genital ridge during embryonic development. Recipient mice containing intratubular injections of testis cell suspensions from C57 mice were allowed to develop for over 1 year, whereupon animals were sacrificed and testis tissue examined by light and electron microscopy. Donor mouse cells formed normal cell associations (stages) as viewed in cross-sectioned tubules. Spermatogonia were found exclusively in the basal compartment, indicating that they were translocated from the tubule lumen through the Sertoli cell junctions, eventually to reside on the basal lamina. Some tubules looked entirely normal from both a quantitative and qualitative standpoint. Others showed qualitative and quantitative impairment. In some tubules a generation of cells was missing from a cell association. A variety of degenerating cells and structural abnormalities were responsible for this impairment, however, the most common abnormalities were seen during the elongation phase of spermatogenesis. Elongation abnormalities and the subsequent degeneration of these cells led to the presence of fewer-than-expected elongate spermatids. There were regions of the testis where no spermatogenesis was noted and only Sertoli cells were present. These regions were generally typical of the testis histology seen in animals not exposed to injected germ cells. However, Sertoli cells in these regions phagocytosed sperm produced in spermatogenically active regions of the tubules. Because transplantation of germ cells, either from fresh or from frozen cells, had wide-ranging implications in biology and medicine, characterization of spermatogonial transplants is an important step in improving this procedure.

The mammalian testis is innervated by extrinsic catecholaminergic nerves and responds to catecholamines with steroid secretion. Although the primate testis has also been shown to be innervated, potential differences in the density of this innervation between immature and sexually developed individuals have not been described. A recent study demonstrated that the primate ovary contains a network of neuron-like cells and that some of these cells are catecholaminergic. It is thus possible that the male gonad is also endowed with a similar intragonadal source of catecholamines. The present study addresses these two issues. Catecholaminergic nerves were identified as such by their content of immunoreactive tyrosine hydroxylase (TH; the rate-limiting step in catecholamine biosynthesis), and in some cases by glyoxylic acid histochemistry. Fibers containing TH were abundant in testes from juvenile animals (1-2 yr of postnatal life), but the density of this innervation was not maintained in adult animals, whose testis showed only a few TH-positive fibers scattered in the interstitial tissue. Testicular norepinephrine (NE) concentration was much lower in adult than in juvenile animals, suggesting that the marked increase in testicular weight that occurs with the attainment of sexual maturity is not accompanied by corresponding changes in NE content. At the ultrastructural level, testicular nerve fibers contained pleiomorphic, dense-core and clear vesicles, suggesting the presence of catecholamines and other neurotransmitters. In addition to this extrinsic catecholaminergic innervation, prepubertal testes, but not adult gonads, contained an intrinsic population of TH-immunopositive neuron-like elements, identified as cells by confocal scanning laser microscopy. To determine whether the prepubertal monkey testis indeed expresses the TH gene, testicular RNA was subjected to reverse transcriptase polymerase chain reaction to amplify the 5' end of TH mRNA, which encodes the regulatory domain of the enzyme. The cDNA that was obtained predicts an amino acid sequence similar, but not identical, to that encoded by the alternatively spliced type 1 TH mRNA form present in the adrenal gland. These results indicate 1) that the primate testis receives a dual catecholaminergic input, one provided by the extrinsic innervation and the other by neuron-like cells located within the gonad itself, and 2) that the influence exerted by both sources on testicular function may be more prominent during the prepubertal period than in adulthood. The presence in the testis of a TH mRNA variant encoding amino acid substitutions in its 5' end suggests that regulation of testicular TH enzyme activity may include a gonad-specific component.

Treatment of male rat pups with the reversible goitrogen 6-n-propyl-2-thiouracil (PTU), administered by adding 0.1% PTU to the mother's drinking water from birth through weaning, increases adult testis size, number of Sertoli cells, and daily sperm production. Adult Leydig cell numbers are also increased by 70%, despite permanent suppression of serum luteinizing hormone (LH). The present study was designed to test whether this effect results from an increase in proliferation of Leydig cells or the mesenchymal precursors. The labeling indices (LI) of six interstitial cell types were measured by tritiated thymidine autoradiography. With the exception of Leydig cells, LI declined after birth for all interstitial cell types in both control and PTU-treated rats, but mesenchymal cell proliferation was not significantly different in control and treated rats. In contrast, the LI of Leydig cells from PTU-treated rats rose by day 10, remained elevated through day 45 (highest on day 35 at 3.6-fold higher than control, P < 0.05), and declined to control values by day 50. The LI of Leydig cells in control rats was unchanged during the experiment. Proliferating Leydig cells were immature, as shown by their cytoplasmic lipid droplets. Adult Leydig cells, which lack lipid droplets, did not proliferate. Mesenchymal and immature Leydig cells contained thyroid hormone receptor mRNA; levels in adult Leydig cells were reduced but detectable. In conclusion, proliferation of Leydig cells, rather than increased proliferation of their mesenchymal precursors, is the principal mechanism responsible for the increase in Leydig cell number after neonatal hypothyroidism. Increased Leydig cell proliferation could result from direct effects of hypothyroidism on these cells. Alternatively, the increased Sertoli cell population that results from PTU treatment could also stimulate increased Leydig cell proliferation, or both of these mechanisms could contribute to the Leydig cell increase.

In line TGR(mRen2)26 transgenic rats (TGR26) bearing a randomly inserted additional renin transgene, the males, but not the females, were found to be infertile. Tissue was obtained from TGR26 males and littermate controls after perfusion fixation, and the morphology of the testes and epididymides was examined. Testis size was normal as was gross morphology, but careful examination revealed that the release of many spermatozoa at stage IX of the spermatogenic cycle was impaired. In addition, the process of cytoplasmic elimination was abnormal, as cytoplasmic fragments of elongate spermatids were present in the epididymis. In TGR26 males, seminiferous tubule lumen size was significantly larger (p < 0.001) than in littermate controls, a difference that was most marked at stages IX-XIV--an effect that could be related to the retention of spermatozoa. In situ hybridization confirmed that expression of renin mRNA could be detected in testes of TGR26 rats but not in normal controls or in a fertile line (TGR27) of rats bearing the same transgene. Immunocytochemistry and in situ and Northern hybridization were used to elucidate the pattern of expression of genes that previous studies have implicated in the process of sperm maturation and/or release. Of the gene products examined (sulphated glycoproteins 1 and 2 [SGP-1, SGP-2], transition proteins 1 and 2 [TP-1, TP-2], urokinase, and cyclic protein 2 [CP-2]), none showed any major change in the pattern of expression compared with that in controls. We postulate that TGR26 transgenic male rats may be infertile because the expression of a gene (or genes) involved in the process of cytoplasmic elimination and/or sperm release has been disrupted by the presence of the transgene close to or within the gene(s). Future planned studies will involve determination of the insertion site(s) and ultrastructural analysis of the final phases of spermiogenesis.

The present study describes the effects of mating on urethral gland acinar cells in male mice. Histological and morphometric analysis demonstrated that there was a depletion of secretory granules in the urethral glands during mating. However, no change occurred in the rough endoplasmic reticulum containing tubular elements. The results indicate that the urethral glands are functional during mating. The timing of their granule depletion suggests that urethral gland secretions may contribute to the formation of semen or the copulation plug.

The surface relationships of the Sertoli cell and the surface relationships of the Sertoli cell in comparison to the changing volumes of developing germ cells were studied using morphometric techniques at periods representing nine groupings of the fourteen defined periods in the cycle of the seminiferous epithelium of the adult rat. No cyclic variation in the total Sertoli plasma membrane surface area was noted. Cyclic variations were noted in the area of the Sertoli cell surface that faces the basal compartment germ cells, but not the basal lamina. No cyclic variations were noted in the amount of contact of the Sertoli cells with each other at the level of the Sertoli cell barrier. However, when areas in the adluminal compartment were studied, significantly less Sertoli-Sertoli contact was seen in stages V through VII than in other stages with the exception of stages II-IV. Surface contact of germ cells with Sertoli cells increased progressively as germ cells entered the intermediate compartment and progressed to late spermatids. However, a calculation of the surface-to-volume ratio showed that surface increases of the Sertoli cell in relation to the volume of germ cells were greatest in elongating spermatids past step 12 of spermiogenesis. The area in which Sertoli ectoplasmic specializations faced germ cells was determined throughout spermatogenesis, and these data demonstrated that the first appearance of ectoplasmic specialization was at the mid-pachytene phase. They also showed that stage VIII was a period when ectoplasmic specialization loss from the cell surface was evident. Less Sertoli ectoplasmic specialization face step 8 and step 19 spermatids than comparable germ cell types at other stages. In addition to Sertoli cell surface area changes during the cycle, volumes of individual germ cell types were determined for the first time. The data presented allow an objective understanding of the complex structure and relationships of the Sertoli cell and provide a basis for understanding functional changes and interpreting biochemical data.

A unique isozyme of angiotensin-converting enzyme (ACE) is produced in large amounts by developing germ cells and is referred to as testis ACE. This protein results from the transcriptional recognition of a sperm-specific promoter during spermatogenesis. The expression within germ cells of testis ACE mRNA was studied using in situ hybridization, ribonuclease (RNase) protection, and Northern analysis. In parallel, testis ACE protein expression was measured through use of Western blot analysis and immunohistochemistry. Although testis ACE protein is first detected in step 10 spermatids, testis ACE mRNA is first detected in a developmentally younger cell population, late pachytene spermatocytes. Thus, testis ACE mRNA is translationally arrested for a period of several days until late in spermiogenesis.

The occurrence and possible functions of mucosal immunity in the male urogenital tract have not been extensively investigated. In this study we used immunolabeling to localize secretory component (SC) and immunoglobulin (Ig) A in the urogenital tract of the male mouse. SC was located in the ventral prostate, while SC and IgA plasma cells were both detected in the urethral glands in the pelvic and bulbous portions of the urethra. SC and IgA were not observed elsewhere in the urogenital tract. We also examined the ventral prostate and urethral glands of sham-castrated, oil-treated castrated, and testosterone-treated castrated mice. There was a striking reduction in the size of the ventral prostate and urethral glands in oil-treated castrates compared to the other two groups, based on gross and histological morphology. Morphometric analysis showed that the cell and nuclear sizes of the urethral gland acinar cells were reduced after castration and restored to normal size by testosterone treatment. Androgen receptors (AR) were localized in the nuclei of urethral gland cells by immunocytochemistry using anti-AR antibodies. Labeling of SC and IgA plasma cells was similar in the urethral glands and ventral prostates of sham- and testosterone-treated castrates, but was reduced or absent at these sites in oil-treated castrates. These studies show that the ventral prostate and urethral glands may be sites for secretory immunity in the male murine urogenital tract, and that the urethral glands are targets for testosterone.

A line of transgenic mice that carries an insertional mutation in a gene essential for spermatogenesis is described. Males homozygous for the transgenic insert are sterile, while female homozygotes and both male and female heterozygotes exhibit normal fertility. Developing spermatids in homozygous males form prominent abnormal multinucleated syncytia (symplasts) and do not complete maturation. In addition, abnormal cytoplasmic vacuolation is commonly seen in Sertoli cells. One flank of the transgenic integration site within the genome has been cloned and used to show linkage between homozygosity for the transgene and the mutant phenotype. The flank maps to mouse chromosome 14 approximately 4 centimorgans proximal to the gene encoding esterase-10 (Es-10). As no other gene that is known to be essential for spermatogenesis has been mapped to this region of the genome and as the mutant phenotype is unique, the transgenic insert appears to affect a previously unidentified gene. We have named the mutation "symplastic spermatids" (sys).

Criteria were devised for determining the elongate spermatid-Sertoli cell ratio in various mammalian species at the electron microscope level. When data from particular species were pooled, the values were: rabbit, 12.17:1, hamster, 10.75:1; gerbil, 10.64:1; rat, 10.32:1; guinea-pig, 10.10:1; vole, 9.75:1; and monkey, 5.94:1. The elongate spermatid-Sertoli cell ratio is a measure of the workload of the Sertoli cell and is a prime factor determining their efficiency. The higher the ratio, the higher the sperm output is likely to be per given weight of seminiferous tubule parenchyma for a particular species.

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.

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.

Cytochalasin D (CD) was used to perturb actin filaments of the Sertoli ectoplasmic specialization (ES)--a cytoskeletal complex of the Sertoli cell related to spermatids. CD (500 microM for 6 h) produced a loss of 88% of the ES facing the head region of early (Step 8) elongating spermatids as compared to vehicle (dimethylsulfoxide:saline) controls. Nitrobenzoxadiazole-phallacidin staining of F-actin revealed a CD-related loss of uniform fluorescence over the head of elongated spermatids. To examine for a possible relationship between the presence of actin and cell attachment at ES sites, hypertonic fixatives were introduced to provoke cell shrinkage and stress ES-associated junctions. After osmotic stress, cell-to-cell adhesion at ES sites remained intact in vehicle-treated animals. CD treatment caused Sertoli cells to separate from elongating spermatids at sites where ES had been lost from the Sertoli cell surface. It is suggested that actin of the ES plays a role in cell-to-cell interaction analogous to its possible role at the Sertoli cell barrier. In CD-treated animals, structures resembling tubulobulbar complexes frequently developed at sites where ES was lost, suggesting that the loss of ES has a facilitatory role in tubulobulbar complex formation. It is hypothesized that tubulobulbar complexes are devices that rid the cells of ES-associated junctional links to effect dissociation of the spermatid from the Sertoli cell during spermiation. Spermatids at Step 8 of development are known to become oriented with their acrosomes facing the base of the Sertoli cell. After CD treatment, a 5.8-fold increase in malorientation of Step 8 spermatids was noted. A role for the ES cytoskeletal complex in orienting the spermatid acrosome toward the basal aspect of the Sertoli cell is also suggested.

A high resolution, two-dimensional (2-D) electrophoretic map of the plasma membrane (PM) polypeptides from the ejaculated boar spermatozoon is described. 2-D silver-stained polyacrylamide gel electrophoresis (PAGE) gels revealed over 250 polypeptides; Coomassie blue staining revealed more than 100. Fifty Coomassie-staining polypeptides were catalogued and biochemically characterized, with twenty of these designated major sperm PM polypeptides. Cavitation pressures ranging from 50 PSI to 1000 PSI were used to enrich 2-D maps either in head PM (50 PSI) or in flagellar PM (1000 PSI) and provided tentative localization of certain PM polypeptides. Immunoabsorption chromatography showed that most major polypeptides seen in the 2-D map were antigenic. Major polypeptide bands from single dimensional (1-D) gels were excised, antibodies against them were produced in rabbits, and the polypeptides were localized via indirect fluorescein isothiocyanate (FITC) technique. Cross-reacting antigenic determinants in the PAGE PM polypeptides were determined by transblotting and staining the transblots by an indirect peroxidase technique. Cross-reactivity was extensive with several polypeptide groups, but specific enough with others to allow tentative localization. Lectin affinity chromatography using Con A, WGA, RCA-1, PNA, and DBA indicated the lectin specificity of PM polypeptides. These data together with periodic acid-Schiff (PAS) and carbohydrate-specific silver staining permitted identification of glycoproteins in the 2-D maps. FITC coupled to specific lectins showed the regional distribution of these lectins on the sperm surface. The 2-D polypeptide map and the catalogue of properties of major Coomassie-stained PM polypeptides provides a reference for future studies in the boar and other species.

This study provides quantitative information on the testes of seasonally breeding golden hamsters during active and regressed states of gonadal activity. Seminiferous tubules occupied 92.5% of testis volume in adult gonadally active animals. Leydig cells constituted 1.4% of the testicular volume. The mean volume of an individual Leydig cell was 1092 microns 3, and each testis contained about 25.4 million Leydig cells. The volume of an average Sertoli cell nucleus during stage VII-VIII of the cycle was 502 microns 3. A gram of hamster testis during the active state of gonadal activity contained 44.5 million Sertoli cells, and the entire testis contained approximately 73.8 million Sertoli cells. Testes of the hamsters exposed to short photoperiods for 12-13 wk displayed a 90% reduction in testis volume that was associated with a decrease in the volume of seminiferous tubules (90.8% reduction), tubular lumena (98.8%), interstitium (72.7%), Leydig cell compartment (79.3%), individual Leydig cells (69.7%), Leydig cell nuclei (50.0%), blood vessels (85.5%), macrophages (68.9%), and Sertoli cell nuclei (34.1%). The diameter (61.1%) and the length (36.8%) of the seminiferous tubules were also decreased. Although the number of Leydig cells per testis was significantly lower (p less than 0.02) after short-photoperiod exposure, the number of Sertoli cells per testis remained unchanged. The individual Sertoli cell in gonadally active hamsters accommodated, on the average, 2.27 pre-leptotene spermatocytes, 2.46 pachytene spermatocytes, and 8.17 round spermatids; the corresponding numbers in the regressed testes were 0.96, 0.20, and 0.04, respectively. The striking differences in the testicular structure between the active and regressed states of gonadal activity follow photoperiod-induced changes in endocrine function and suggest that the golden hamster may be used as a model to study structure-function relationships in the testis.

Culturing Sertoli cells has proven to be a valuable technique to study the responses of these cells under a variety of conditions. This minireview focuses on the culture system and how it has contributed to our knowledge of the capabilities of isolated Sertoli cells. In addition, it focuses on the limitations of the system and the gaps in our knowledge about Sertoli cells in culture and what the results of in vitro studies mean in the intact animal. Caution is urged in interpreting in vitro results to the intact animal without their validation in vivo.

Caput and cauda epididymal fluids were found to be exceedingly rich in the numbers and kinds of polypeptides when analyzed by two-dimensional (2-D) gel electrophoresis. Only a few of the major (Coomassie-stained) and minor (silver-stained) epididymal fluid polypeptides were identified on epididymal sperm plasma membranes (PM) and even fewer identified in ejaculated sperm. The 2-D electrophoretic patterns of caput sperm PM differed little from those of cauda sperm PM. Thus, epididymal transit resulted in relatively minor quantitative and qualitative modifications in sperm PM composition. Seminal plasma showed a few major polypeptides from the cauda epididymal fluid, but the major constituents were those polypeptides from the seminal vesicle secretions. Sperm appear to acquire one acidic high molecular weight polypeptide from either the bulbourethral gland or prostate gland, and another major acidic polypeptide of high molecular weight from the seminal vesicle gland. Numerous neutral and basic low molecular weight polypeptides, originating from the seminal vesicles, adhered tightly to sperm. These were major polypeptides and constituted a substantial percentage of the total PM protein. Thus, major contributions to the sperm PM polypeptide profile occurred at ejaculation. This study did not address loosely bound polypeptides but is the first to analyze, in a comprehensive way, the origins of tightly bound sperm polypeptides from a single species.

Properties of (Ca2+ + Mg2+) adenosine triphosphatase (ATPase) in plasma membranes from boar epididymal spermatozoa are described. Enzyme activity is optimum at high pH and has a high affinity for Ca2+. It is not inhibited by the calmodulin antagonist trifluoperazine (TFP), but it is inhibited by low concentrations of Ca2+. Plasma membrane vesicles obtained by hypotonic lysis of intact sperm [mixed inside-out (IOV) and right side-out (ROV) vesicles] transport 45Ca2+ in the presence of oxalate. Similar to the Ca2+-stimulated Mg ATPase activity, transport is unaffected by TFP, but unlike the ATPase, transport is at an optimum rate near neutral pH and is completely inhibited by p-chloromercurphenylsulfonate (pCMS). When plasma membranes are labeled in the presence and absence of Ca2+ and Mg2+ with [gamma-32P]ATP, differences in the intensity of labeling and lability of bound 32P to alkali and hydroxylamine suggest that two polypeptides between 100-120K may be related to a transport ATPase. The addition of TFP at concentrations which stimulate net Ca2+ uptake in intact cells causes intense labeling of a single neutrally charged protein near 68K. These labeling patterns and the properties of (Ca2+ + Mg2+) ATPase identify particular plasma membrane proteins (PMPs) from the complex surface of these cells that may be involved in Ca2+-dependent functions and support the view that calmodulin is not directly involved in the regulation of ATP-driven Ca2+ efflux from boar spermatozoa.

Fertilization experiments using zona-free hamster eggs and spermatozoa from both guinea pig and human were conducted in the presence of cytochalasin D to evaluate the possible role of actin filaments in fertilization processes. When the actin filament inhibitor, cytochalasin D, was added to fertilization media at concentrations of 10 to 30 microM, penetration of eggs was significantly inhibited. Preincubation of the eggs with cytochalasin D and washing prior to addition of spermatozoa had no effect on penetration as quantitated by the number of swollen heads in the egg cytoplasm. However, spermatozoa preincubated with cytochalasin D and subsequently washed prior to egg addition showed reduced penetration of the same magnitude as when spermatozoa and eggs were coincubated with cytochalasin D. Both the percentage of zona-free eggs showing decondensed sperm heads and the penetration indices (total decondensed spermatozoa/total eggs) were significantly affected when spermatozoa were exposed to cytochalasin D. The DMSO vehicle used to dissolve cytochalasin D had little effect on the number of decondensed heads. When the concentration of cytochalasin D was increased (DMSO remaining constant) in human sperm experiments, percent penetration decreased and progressively fewer decondensed spermatozoa were recorded, indicating dose-responsiveness to cytochalasin D. Motility parameters of human spermatozoa were not altered at any of the concentrations of cytochalasin D tested. Neither guinea pig sperm motility nor acrosome reaction was altered significantly by cytochalasin D or the DMSO vehicle. These experiments suggest that cytochalasin D may be an inhibitor of some fertilization processes such as sperm penetration or sperm head decondensation.