Modulation of basal and FSH-dependent cyclic AMP production in rat seminiferous tubules staged by an improved transillumination technique

The stage-dependent action of follicle-stimulating hormone (FSH) in the rat seminiferous epithelium was investigated in microdissected 1 mm tubule segments, where the precise stage of the cycle was identified by a rapid screening method of live cell squash preparations. For distinction of stages I and II and the substages of VII, new criteria were used. The step 16 spermatids with rapid assembly of outer dense fibers leading to marked increase of flagellar thickness were used for distinction of stages I and II. The form and density of the cytoplasmic lobes of step 19 spermatids was used for recognition of substages of VII. Highest basal production of cyclic AMP (cAMP, measured by radioimmunoassay) was found in stage II of the cycle and stages XIV-I-VI had higher values than did stages VII-XIII. A decline occurred during stage VII and an increase at stage XIV. When stimulated with FSH, highest cAMP secretion was found in stage IV of the cycle; again, stages XIV-I-VI had higher values than did other stages. A small but significant (P less than .01) stimulation was found at substage VIId. FSH-stimulated and basal cAMP productions of different stages were compared, highest values were found at stages IV and XIII, and lowest, at stages VIIa-c and IX of the cycle. Since the FSH-dependent cAMP production is confined to Sertoli cells, and the number of these cells is constant per unit length of seminiferous tubules, the Sertoli cells are obviously under a stage-specific paracrine control by the surrounding spermatogenic cells. Specific steps in cell differentiation, such as spermatogonial proliferation, final maturation of the spermatids (stages I-VII), onset of meiosis (substage VIId), and completion of meiotic divisions (stage XIV) may be involved in this interaction.     

Degeneration of germ cells in normal,hypophysectomized and hormone treated hypophysectomized rats

In normal adult rats some germ cells degenerate at several vulnerable steps of spermatogenesis. These are the type A spermatogonia, midpachytene spermatocytes, primary and secondary spermatocytes which degenerate during their respective maturation divisions and step 7 and 19 spermatids. In the present study, these degenerating cells were examined under the electron microscope, and their frequency was determined in toluidine blue stained semithin sections of testes from normal, hypophysectomized (at 5.5 days after operation) and hypophysectomized rats injected with FSH and LH separately or in combination. With the exception of the step 19 spermatids, the degenerating germ cells underwent necrosis in vacuolated spaces delimited by Sertoli cells. In the case of the affected step 19 spermatids, an apical cytoplasmic process of the Sertoli cell initially ensheathed a long segment of their flagellum, and then each degenerating cell was drawn deep in the seminiferous epithelium where it was phagocytozed by the Sertoli cell. Soon after hypophysectomy the incidence of degenerating mid-pachytene spermatocytes, step 7 and 19 spermatids which are present in stages VII or VIII of the cycle of the seminiferous epithelium, increased significantly. In contrast the number of degenerating primary or secondary spermatocytes during the meiotic divisions seen in stage XIV of the cycle or of any other germinal cell was not significantly modified. While the injection of FSH alone had no influence on the number of degenerating cells in hypophysectomized rats, injections of LH at the two doses administered (0.7 μg or 20 μg) reduced significantly the number of degenerating cells seen in stages VII-VIII of the cycle; combined injections of FSH and LH (20 μg) reduced the number of these degenerating cells to the normal low values. Thus it appeared that the mid-pachytene spermatocytes and the step 7 and 19 spermatids, all present in the adluminal compartment of the seminiferous epithelium in stages VII or VIII of the cycle, were more sensitive to the presence of absence of gonadotropic hormones than the other germ cells present in the seminiferous epithelium.     

Use of seminiferous tubule segments to study stage specificity of unscheduled DNA synthesis in rat spermatogenic cells

DNA repair in spermatogenic cells at various stages of maturity was determined by quantitation of unscheduled DNA synthesis (UDS). Male F-344 rats were exposed (i.p.) to methyl methanesulfonate (MMS, 35 mg/kg); 1 hr later, segments of seminiferous tubules corresponding to spermatogenesis stages II, IV-V, VI, VII, VIII, IX-X, XII, and XIV were isolated with the transillumination pattern of the tubules as a guide. Intact tubule segments were cultured 24 hr in the presence of [3H]thymidine, and UDS was quantitated by autoradiography as net grains/nucleus (NG). In primary spermatocytes from treated rats, NG count increased with increasing maturity from leptotene primary spermatocytes (3.5 NG) up through stage VIII and IX-X pachytene spermatocytes (22 NG), after which NG decreased in stage-XII pachytene and diplotene spermatocytes (to 16 NG and 8 NG, respectively). Round spermatids of steps 2-8 of spermiogenesis all exhibited approximately the same UDS response (8 NG). Elongating spermatids as mature as step 14 underwent UDS after exposure to MMS, but step-15 and later-step spermatids did not. The DNA repair response of pachytene spermatocytes cultured within segments of seminiferous tubule corresponding to stages VIII and IX-X was 4 to 25 times greater, depending on the dose of MMS, than pachytene spermatocytes isolated by enzymatic digestion and cultured in suspension [Bentley and Working, Mutat Res 203:135-142, 1988]. Thus, the use of segments of seminiferous tubule both increased the sensitivity of UDS as an indicator of DNA damage in rat germ cells and enabled the study of UDS in spermatogenic cells at different stages of maturity.     

Morphometric studies on rat seminiferous tubules

The goal of this morphometric study was to obtain quantitative information on the seminiferous tubules of Sprague-Dawley rats, including changes seen at various stages of the cycle of the seminiferous epithelium. Tissue from perfusion-fixed testes was embedded in Epon-Araldite; and sections were subjected to morphometric measurements at the light microscopic level, using point counting for volume densities and the Floderus equation for numerical densities. Changes occur in the diameter of the seminiferous tubule, as well as in the volume of the seminiferous epithelium and tubule lumen, from stage to stage during the cycle. A significant constriction of the seminiferous tubule accompanies spermiation. The volume of the seminiferous epithelium per unit length of the tubule begins to increase after stage XIV, and peaks at stage V of the next cycle. The tubule lumen increases dramatically from stages V to VII, at the expense of the epithelium. The number of Sertoli cells is constant per unit length of the seminiferous tubule at all stages of the cycle. This is also true for primary spermatocytes of various developmental phases and for round spermatids from step 1 through step 10 of spermiogenesis. The average number of younger (preleptotene, leptotene, zytgotene) primary spermatocytes per Sertoli cell is 2.34 ± 0.082 (SEM), the number of older (pachytene, diplotene) primary spermatocytes per Sertoli cell is 2.37 ± 0.064, and the ratio of step 1–10 spermatids to Sertoli cells is 7.89 ± 0.27. By studying tangential views of serially sectioned seminiferous tubules at stage V, it is shown that the number of step-17 spermatids associated with each Sertoli cell averages 8.35 ± 0.128, although the counts ranged from 6 to 11. The only appreciable occurrence of cell death after the last spermatogonial mitosis appears to be a 15% loss during the first meiotic division. From our morphometric results, corrected for volume changes during preparation for microscopy, there are 15.7 million (± 0.99 million) Sertoli cells per gram of fresh rat testis. The length of seminiferous tubule per gram of testis is estimated to be 12.4 ± 0.56 meters, and the tubule surface area per gram testis is 119.7 ± 2.57 cm². The daily production of mature spermatids is 9.61 million (± 0.615 million) per gram of testis.     

Surface interaction in vitro between sertoli cells and germ cells at different stages of spermatogenesis

The presence of surface-recognition mechanisms between somatic and germ cells of the seminiferous epithelium has been studied in the rat by an assay in vitro based on the ability of homogeneous populations of spermatogenic cells, at specific differentiative stages, to adhere to monolayers of cultured Sertoli cells. The results show that germ cells adhere specifically to Sertoli cells and that the adhesion is dependent on the differentiative stage of the germ cells. Pachytene spermatocytes show the highest ability to adhere and form typical junctional specializations with the underlying Sertoli cells, while round spermatids adhere much less to the substrate. The possible regulative role of a somatic cell-germ cell interaction is discussed.     

Seminiferous tubule stages in the prairie dog (Cynomys ludovicianus) during the annual breeding cycle

The male prairie dog (Cynomys ludovicianus) is an annual breeder with complete testicular regression between breeding periods. Knowledge of the seminiferous tubule cycle stages at all phases of the annual cycle is essential for evaluation of testicular effects of endogenous and exogenous hormones. Testis tubule diameter is directly correlated with testicular weight during the annual cycle. Seminiferous tubule stages found during testicular activity start with sperm release and round spermatids in the Golgi stage (I). Then they progress through the cap and acrosome stages (stages II to VI) until elongate spermatids are formed. During these stages preleptotene, leptotene and zygotene cells develop into pachytene cells which mature with the long spermatids (stage VII). Two distinct tubule associations (stages VIII, IX) follow during which the first and second meiotic metaphases occur. These stages are correlated with the middle and late phases of residual lobe retraction and condensation. The last stage (X) has final sperm development and is present with round spermatids that have no Golgi development. During regression changes are initially associated with the seminiferous tubule stages of active testes and end with relocation of Sertoli cell nuclei to a position above the basal layer of spermatogonia. Out of season testes are characterized by few spermatogonial mitoses and absence of viable spermatocytes. In recrudescent testes, Sertoli cell nuclei again become basal, spermatogonia resume mitoses and spermatocytes and spermatids progressively develop. After each cycle of proliferation of germ cells there is sloughing of the most differentiated spermatocytes and spermatids until the final tubule associations of the active testis are present. Anat. Rec. 247:355–367, 1997. © 1997 Wiley-Liss, Inc.     

Spermatogenesis in the dog

The cycle of the seminiferous epithelium of the dog was divided into eight stages, using as criteria the shape of the spermatid nucleus, the location of spermatids and spermatozoa in regard to the basement membrane, the presence of meiotic figures and the release of spermatozoa from the lumen of the tubule. Based upon these criteria, a modification of the eight-stage system of classification of the cycle of the seminiferous epithelium was developed. Cell populations making up each stage are described. The relative frequencies of stages 1 through 8 were 21.9, 12.7, 2.8, 11.5, 8.3, 15.4, 13.3 and 14.0%, respectively. The duration of one cycle of the seminiferous epithelium was 13.6 days (SE ± 0.7), as determined from cells labeled by tritiated thymidine. The absolute durations of stages 1 through 8 were 3.0, 1.7, 0.4, 1.6, 1.1, 2.1, 1.8 and 1.9 days, respectively. The life span of primary spermatocytes was 20.9 days, of secondary spermatocytes 0.5 days, spermatids with round nuclei 10.5 days, spermatids with elongated nuclei up to the time they are released into the lumen, 10.6 days. Counts of the different types of spermatogenic cells in tubular cross sections revealed little or no germ cell degeneration during the two maturation divisions.     

Relative volume of sertoli cells in monkey seminiferous epithelium: A stereological analysis

Techniques of quantitative stereology have been utilized to determine the relative volume occupied by the Sertoli cells and germ cells in two particular stages (I and VII) of the cycle of the seminiferous epithelium. Sertoli cell volume ranged from 24% in stage I of the cycle to 32% in stage VII. Early germ cells occupied 3.4% in stage I (spermatogonia) and 8.7% in stage VII (spermatogonia and preleptotene spermatocytes). Pachytene spermatocytes occupied 15% (stage I) and 24% (stage VII) of the total volume of the seminiferous epithelium. In stage I the two generations of spermatids comprised 58% of the total epithelium by volume, whereas in stage VII, after spermiation, the acrosome phase spermatids occupied 35% of the total seminiferous epithelial volume.     

DOG1 immunohistochemical staining of testicular biopsies is a reliable tool for objective assessment of infertility

Testicular biopsy may be a component of the work-up of male infertility. However, no reliable diagnostic tools are available for objective quantitative assessment of spermatogenic cells. It is well known that MAGE-A4 is selectively expressed in spermatogonia and our group has previously demonstrated that DOG1 differentially stains germ cells. Therefore, we performed DOG1 and a double stain cocktail (DOG1 and 57b murine monoclonal anti-MAGE-A4) immunohistochemical stains on 40 testicular infertility biopsies (10 each with active spermatogenesis, Sertoli cell-only, hypospermatogenesis, and maturation arrest), 25 benign seminiferous tubules from radical orchiectomies, and 5 spermatocytic tumors (ST). In biopsies/resections with active spermatogenesis, DOG1 stained spermatocytes and spermatids and was absent in spermatogonia, while MAGE-A4 stained spermatogonia and primary spermatocytes (weak). In hypospermatogenesis, DOG1 highlighted decreased spermatocytes/spermatids and MAGE-A4 highlighted decreased spermatogonia. DOG1 staining confirmed decreased to absent spermatocytes in maturation arrest and MAGE-A4 staining established the presence of preserved spermatogonia in all cases. All STs were negative for DOG1 and positive for MAGE-A4, while all Sertoli cell-only cases were negative for DOG1 and the double stain cocktail. In conclusion, we confirmed that DOG1 is expressed in spermatocytes and spermatids and MAGE-A4 highlights primarily spermatogonia. Usage of these stains facilitates confirmation of maturation arrest, assessment of the percentage of testis involvement in hypospermatogenesis and identification of mixed patterns. Finally, this study supports that the differentiation of STs is more closely related to spermatogonia than the more mature spermatocytes.     

Correlation between blood-testis barrier development and onset of the first spermatogenic wave in normal and in busulfan-treated rats: a lanthanum and freeze-fracture study

Pregnant rats (day 13) received 10 mg/kg of Busulfan i.p. The seminiferous tubules of their offspring from post-natal age 1 day up to day 35 were examined with TEM after fixation plus intercellular tracers, and with freeze-fracture techniques. During this period, the inter-Sertoli tight junctions of controls increase both in number and in length. Between days 10 and 13 the seminiferous cords have numerous preleptotene and leptotene spermatocytes surrounded by tracer. The inter-Sertoli junctions are tortuous and predominantly perpendicular to the basal lamina. Between ages 13 and 20 days the seminiferous epithelium reaches zygotene-pachytene stages. The tracer is stopped at the inter-Sertoli junctions at this stage, whereas it still permeates tubules displaying preleptotene and leptotene spermatocytes. Freeze-fracture shows that the orientation of inter-Sertoli junctions has changed to parallel, both to each other and to the basal lamina. In the Busulfan-treated rats, the tubules continue having, up to post-natal day 30, only Sertoli cells and scanty spermatogonia. In these, lanthanum penetration goes as far as the apical Sertoli cell region; the inter-Sertoli junctions still show tortuous strands, and most are oriented perpendicular to the basal lamina. This indicates that formation of the first competent inter-Sertoli junctions is temporo-spatially simultaneous with the appearance of zygotene-pachytene spermatocytes.     

Hypospermatogenesis is the Cause of Infertility in the Male Weaver Mutant Mouse

The pathologic phenotype of the testis in both prepuberal and postpuberal male weaver mutant mice was studied by light microscopy. Morphometric analysis of seminiferous tubules was carried out. Epididymal fluid was examined for the presence of spermatozoa. The seminiferous tubules of 21-day-old prepuberal weaver mutant mice lacked patent lumina and had more degenerated cells than control mice. Fifty-six day-old weaver mutants had many germinal epithelial cells located within the adluminal compartment that were in advanced stages of degeneration. Round spermatids were enlarged and multinucleated. Round spermatids and spermatocytes had sloughed into the lumen. Compared to control mice, elongated spermatids were seen less frequently. In older weaver mice, the degenerative process involved germ cells in both the adluminal and basal compartments. In 143- and 226- day-old weaver mutants, the Sertoli cells were atrophic. Diameters of seminiferous tubules in weaver mice were significantly reduced when compared to control mice. Sperm were either absent or very low in number in the epididymal fluid of postpuberal weaver mice. We conclude that spermatogenesis is abnormal in male weaver mutant mice. The testicular phenotype is characterized by a degenerative process that affects both germ cells and supporting cells.     

Role of the spermatogenic–Sertoli cell interaction through cell adhesion molecule-1 (CADM1) in spermatogenesis

Endocrine and local secretory factors have long been known to be required for spermatogenesis. Evidence has been accumulating in recent years indicating that direct contact between spermatogenic and Sertoli cells is also required for spermatogenesis. Cell adhesion molecules of various types have been found in the mammalian testis that are expressed in spermatogenic and/or Sertoli cells and involved in homophilic and/or heterophilic binding. We have cloned a novel cell adhesion molecule, cell adhesion molecule-1 (CADM1), also known as immunoglobulin superfamily 4A or spermatogenic immunoglobulin superfamily, from the mouse testis. CADM1 belongs to the immunoglobulin superfamily and is composed of three immunoglobulin-like domains, a transmembrane domain, and a short intracellular domain. In the seminiferous epithelium, CADM1 is expressed in intermediate spermatogonia through to early pachytene spermatocytes as well as in elongating spermatids—but not in round spermatids, mature spermatozoa, or Sertoli cells. One of the heterophilic binding partners of CADM1 has proven to be a poliovirus receptor, another member of the immunoglobulin superfamily that is expressed in Sertoli cells. Knockout mice for CADM1 develop male infertility due to defective spermatogenesis. These findings suggest that cell adhesion molecules between spermatogenic and Sertoli cells play essential roles in spermatogenesis.     

Endorphin suppresses FSH-stimulated proliferation of isolated neonatal Sertoli cells by a pertussis toxin-sensitive mechanism

During perinatal development, when the size of the Sertoli cell population is determined, Leydig cells produce beta-endorphin, a peptide which may interact with Sertoli cells to modify their FSH-responsiveness, as suggested by our previous work. The goal of the present study was first, to test directly the possibility that beta-endorphin modifies the proliferative response of neonatal Sertoli cells to FSH, and second, to gain information on a mechanism(s) involved in any observed effect. We treated isolated 6-day-old Sertoli cells with FSH or vehicle in vitro and measured their incorporation of exogenous, radiolabeled thymidine with quantitative autoradiography. After 2 days in culture with FSH, we detected a 10-fold increase in the rate of Sertoli cell proliferation. The level of cell division in these FSH-treated cultures was identical to that in other cultures exposed to cAMP under similar conditions. In addition, inclusion of beta-endorphin 3 hr prior to FSH or cAMP decreased the effect of the hormone by 50% but left the cAMP response unchanged. Thus, beta-endorphin acts on isolated, neonatal Sertoli cells at a point prior to intracellular production of cAMP to suppress their response to FSH. When other cultures were treated with pertussis toxin, a blocker of intracellular GTP-binding proteins such as Gi, before sequential addition of endorphin and FSH, the effect of beta-endorphin on FSH-responsiveness was abolished. Moreover, when other cultures were exposed to pertussis toxin in the absence of endorphin, followed by FSH, their response to the hormone was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Movement of spermatocytes from the basal to the adluminal compartment of the rat testis

The progressive movement of primary spermatocytes from the basal to the adluminal compartment of the seminiferous tubule was studies after testes were fixed with standard and hypertonic solutions. In stages VI, VII and VIII of the cycle (classification of Leblond and Clermont, '52), preleptotene spermatocytes were observed within the basal compartment of the seminiferous tubule. Resting on the basal lamina, these cells were bound tightly to neighboring Sertoli cells by desmosome-like junctions. In late stage VIII and early stage IX, basal processes of Sertoli cells were observed between the newly formed leptotene cells and the basal lamina, and in stage IX, the Sertoli processes met to form a junction of the zonula adherens type. This junction formed a permeability barrier which restricted the free access of fixative into the spaces around leptotene cells. Evidence for this was found in the absence of the shrinkage artifact produced with hypertonic solutions in earlier stages. In longitudinal sections, the permeability barrier was first observed in an area of the tubule in which sperm release was also taking place. In mid- stage IX and in stage X, Sertoli-Sertoli junctional specializations formed de novo below the leptotene spermatocyte, while those from the preceding stages, present above the leptotene spermatocytes, remained intact. Thus, tight junctions were in evidence for a considerable period of the time, both above and below the leptotene spermatocytes. At no time in the process of germ cell movement toward the lumen did these cells exhibit evidence of amoeboid movement or lose desmosome-like contacts with the surrounding Sertoli cells. From this study it is concluded that the Sertoli cells play an active role in the transfer of spermatocytes to the adluminal compartment. A transient intermediate compartment of the seminiferous tubule is described, one which allows for the continual maintenance of the blood-testis barrier during transit of spermatocytes from the basal to the adluminal compartment.     

Inhibin B plasma concentrations in oligozoospermic subjects before and after therapy with follicle stimulating hormone

The aim of this study was to investigate inhibin B and follicle stimulating hormone (FSH) secretion in a large group of oligozoospermic subjects affected by different degrees of testicular damage, before and after FSH treatment. A total of 135 oligozoospermic subjects (sperm count < 20 x 10(6)/ml) were evaluated for seminal parameters and FSH, luteinizing hormone (LH), testosterone and inhibin B plasma concentrations. Testicular structure was analysed with bilateral fine needle aspiration cytology. Inhibin B showed an inverse correlation with FSH, no correlation with sperm concentration and a significant relationship with intratesticular spermatid number, demonstrating that testicular spermatids play an important role in the control of inhibin B production. Twenty-five subjects with sperm counts < 10 x 10(6)/ml were treated with FSH; 11 of these had basal FSH and inhibin B plasma concentrations in the normal range (group A), while in seven subjects FSH was elevated (> 7 IU/l) with normal inhibin B (group B), and in seven patients FSH was high and inhibin B reduced (< 80 pg/ml) (group C). During treatment, in group A patients inhibin B plasma concentrations increased significantly after 2, 3 and 4 weeks of FSH administration and declined thereafter to pre-treatment concentrations. Groups B and C did not show any modification during the treatment. In the same period, in group A FSH increased significantly after 2, 3 and 4 weeks and subsequently declined. In groups B and C, FSH increased significantly after 2 weeks and remained elevated during the following period. The results of the present study confirm the significant inverse correlation between inhibin B and FSH plasma concentrations in subjects with disturbed spermatogenesis, and demonstrate that inhibin B reflects Sertoli cell function and their interaction with spermatids. FSH and inhibin B concentrations are an expression of the spermatogenic status of seminiferous tubules. FSH treatment seems to modify inhibin B plasma concentrations only in subjects with normal basal FSH and inhibin B, independently from the effects of this therapy on sperm production.     

Effects of testosterone enanthate on the structure of the male reproductive tract of the rat

The histology and fine structure of the testis, epididymis and sex accessory glands were studied in young adult male rats administered testosterone enanthate, 120 μg/100 g body weight, three times weekly for 4, 8, or 12 weeks. The weights of the testis and epididymis decreased, and animals treated for 11 weeks were infertile. Alterations were found in the seminiferous tubules of all rats treated for 8 or 12 weeks, including the presence of many degenerating germ cells and a-large decrease or absence of late spermatids. Study of different stages of the cycle of the seminiferous epithelium showed that the greatest number of degenerating germ cells, step 7 spermatids and pachytene primary spermatocytes, occurred at stages VII-VIII of the cycle. Some normal appearing spermatogonia, primary spermatocytes and early spermatids remained in most seminiferous tubules. Sertoli cells contained many lipid droplets and lysosome-like bodies, and degenerating cells were surrounded by Ser-toli cell cytoplasm. The Leydig cells of treated animals were greatly reduced in size. Sperm progressively disappeared from the lumen of the middle segment and proximal part of the terminal segment of the epididymis after treatment for 8 or 12 weeks. Changes in the middle segment also included the appearance of intraepithelial cavities containing debris, and the presence within the epithelium of phagocytic cells that resembled leukocytes. The lumen of the proximal part of the terminal segment was often collapsed, while in the distal part of the terminal segment, the lumen was filled with cellular debris and degenerating sperm. Organelles of the principal cells of the epididymal epithelium appeared to be qualitatively unaltered. The weight of the sex accessory glands remained close to normal, and the presence of normal ultrastructural features suggested that production of secretions continued.     

The blood–testis barrier in the toad (Bufo arenarum hensel): A freeze-fracture and lanthanum tracer study

Intercellular junctions between Sertoli cells in the toad testis were studied by freeze-fracture and electron-opaque intercellular markers. These junctional specializations are characterized in thin sections by a series of focal fusions on the outer leaflets of both adjacent cell plasmalemmas, associated with bundles of fine filaments in the subjacent Sertoli cell cytoplasms. However, the wide subsurface cisterna of the endoplasmic reticulum, a component constantly associated with Sertoli cell junctions in mammals, is absent in the toad. The intravascularly injected lanthanum hydroxide, used as a tracer compound, gains access to the seminiferous tubules and surrounds spermatogonia and leptotene spermatocytes, but is persistently excluded from germ cells in later stages of development. This indicates that, as is the case in the mammalian testis, a permeability barrier to lanthanum is established which isolates all germ cells beyond leptotene spermatocytes. Freeze-fracture reveals the characteristic occluding junctions between Sertoli cells, but a variation in their geometric patterns was clearly observed in different regions of the toad seminiferous epithelium. The membrane-fractured faces of Sertoli cells embracing differentiating spermatids exhibit a deep junctional complex: up to 50 rows of particles between adjacent Sertoli cells separate these late germ cells from the periphery of the seminiferous tubules. Sertoli cells surrounding early germ cells generally exhibit, instead, a discontinuous, poorly developed network of interconnected rows of particles with few widely spaced strands. This seems to permit the percolation of the intercellular marker in areas of the seminiferous epithelium containing spermatogonia and leptotene spermatocytes.     

Quantitative aspects of water buffalo (Bubalus bubalis) spermatogenesis.

In the buffalo, seminiferous tubules occupy about 82% of the testis. Spermatogenesis can be divided into 6 stages according to characteristic cellular associations in the seminiferous epithelium. A-spermatogonia have a volume of approximately 1,400 microns3 and the highest absolute mitochondrial volume of all spermatogenic cells. B-spermatogonia display cellular, nuclear and mitochondrial volumes of approximately half the values of A-spermatogonia. From preleptotene (approximately 470 microns3) to late diplotene (approximately 2,300 microns3), the volume* of primary spermatocytes increases nearly five-fold; their nuclear volumes increase by 3.5 times within the same period. During zygotene mitochondrial cristae start to dilate. Grouping of mitochondria by a dense intermitochondrial substance is most prominent during pachytene and diplotene. In pachytene the absolute size of the Golgi apparatus more than doubles, indicating a high secretory activity. Through zygotene only rER is encountered; in pachytene and diplotene a tubular sER makes its first appearance. Secondary spermatocytes are found only in stage 4 of the cycle. Due to partial cell necrosis and autolytic events, late maturation phase spermatids display no more than 25% of the size of cap phase spermatids. There is no morphological evidence for an active uptake and digestion of residual bodies by the Sertoli cells. Also, no lipid cycle is present in the buffalo seminiferous epithelium. Morphometric evaluations reveal that 63% of all theoretically possible germ cells disappear from the seminiferous epithelium during spermatogenesis. Heavy cell loss is observed in stage 4 of the cycle in the spermatogonial fraction as well as during the second meiotic division.     

The blood-testis barrier in the toad (Bufo arenarum Hensel): a freeze-fracture and lanthanum tracer study

Intercellular junctions between Sertoli cells in the toad testis were studied by freeze-fracture and electron-opaque intercellular markers. These junctional specializations are characterized in thin sections by a series of focal fusions on the outer leaflets of both adjacent cell plasmalemmas, associated with bundles of fine filaments in the subjacent Sertoli cell cytoplasms. However, the wide subsurface cisterna of the endoplasmic reticulum, a component constantly associated with Sertoli cell junctions in mammals, is absent in the toad. The intravascularly injected lanthanum hydroxide, used as a tracer compound, gains access to the seminiferous tubules and surrounds spermatogonia and leptotene spermatocytes, but is persistently excluded from germ cells in later stages of development. This indicates that, as is the case in the mammalian testis, a permeability barrier to lanthanum is established which isolates all germ cells beyond leptotene spermatocytes. Freeze-fracture reveals the characteristic occluding junctions between Sertoli cells, but a variation in their geometric patterns was clearly observed in different regions of the toad seminiferous epithelium. The membrane-fractured faces of Sertoli cells embracing differentiating spermatids exhibit a deep junctional complex: up to 50 rows of particles between adjacent Sertoli cells separate these late germ cells from the periphery of the seminiferous tubules. Sertoli cells surrounding early germ cells generally exhibit, instead, a discontinuous, poorly developed network of interconnected rows of particles with few widely spaced strands. This seems to permit the percolation of the intercellular marker in areas of the seminiferous epithelium containing spermatogonia and leptotene spermatocytes.     

Evaluating the anti-fertility potential of α-chlorohydrin on testis and spermatozoa in the adult male wild Indian house rat (Rattus rattus)

To examine the effects of α-chlorohydrin on testis and cauda epididymis in the male house rat (Rattus rattus), 24 adult male rats were segregated into two groups. Group I rats were force-fed daily by intragastric intubation with α-chlorohydrin at a single dose of 1.0 mg/100 g body weight/d for 5, 15, and 45 days. Another group was fed with distilled water, which served as the control. The treated male rats were paired with 24 adult proestrus female rats for 5 days after the last oral treatment and fertility was tested. At the end of the experiments, all of the male rats were weighed and killed by cervical dislocation. The right testes were removed, weighed, and processed for ultrastructural changes of spermatozoa from the cauda epididymis and testis under scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The seminiferous tubular area, nuclear diameter of the Sertoli and Leydig cells, percentage of spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, spermatozoa, and Sertoli cells in each group were compared morphometrically. Our results showed that the percentages of primary spermatocytes steadily increased from 5 to 15 days, but primary and secondary spermatocytes decreased significantly at 45 days. There was a steady decline in the percentages of spermatozoa and spermatids at all fixation intervals in the treated animals, but the percentages of spermatogonia and Sertoli cells increased significantly at 15 and 45 days. Seminiferous tubular areas, nuclear diameter of Leydig and Sertoli cells, and fertility rates were reduced after 45 days of treatment. SEM and TEM studies revealed severe morphological abnormalities in the spermatozoa, including deglutination of the acrosomal part, loss of head capsules, and fragmentation of tail fibrils. There was an enhanced anti-fertility effect and a lower number of implantation sites in the rats treated for 5 days. Our results validate α-chlorohydrin as a successful anti-fertility agent that prevents spermatogenesis.