Interrelationships between Sertoli cells and germ cells in the Syrian hamster

The interrelationships of the Sertoli cells and germ cells in the Syrian hamster were examined using the electron microscope. Demosome-like junctions were observed attaching Sertoli cells to spermatogonia and spermatocytes. In the region of the junctions dense plaques lay on the cytoplasmic surfaces of the plasmalemma of the opposing cells. Sertoli cell cytoplasmic filaments converged in the area of the junctions and inserted into the subsurface densities. Filaments were not observed associated with the subsurface densities of the germ cells. In the region of the junctions a 15...20 nm gap, filled with an attenuate amorphous substance, separated the plasmalemmata. Another attachment device termed "junctional specialization" occurred between Sertoli cells, and preleptotene spermatocytes and all successive developmental steps in the germ cell line in the hamster. The junctional specializations consisted of a mantel of Sertoli cell cytoplasmic filament lying subjacent to the Sertoli cell plasmalemma and an opposed cisterna of the endoplasmic reticulum. In stages VII-VIII preleptotene supermatocytes were observed in transit from the basal compartment to the adluminal compartment. While Sertoli-Sertoli junctions adluminal to the spermatocytes remained intact, typical Sertoli-Sertoli junctions formed between opposed Sertoli cell processes basal to the spermatocytes. It is proposed that, during the passage of spermatocytes in to the adluminal compartment, junctional specializations associated with preleptotene spermatocytes in the basal compartment migrate basal to the spermatocytes and contribute to formation of Sertoli-Sertoli junctions. Treatment of seminiferous tubules with hypertonic media was used to demonstrate that the junctional specializations function in cell-to-cell adhesion. Data indicated that these junctions function to retain the developing spermatids within the seminiferous epithelijm until the time of spermiation. At spermination the junctional specializations disappear and the spermatids drift off into the tubule lumen.     

Oligo-astheno-teratozoospermia in mice lacking RA175/TSLC1/SynCAM/IGSF4A, a cell adhesion molecule in the immunoglobulin superfamily

RA175/TSLC1/SynCAM/IGSF4A (RA175), a member of the immunoglobulin superfamily with Ca2+-independent homophilic trans-cell adhesion activity, participates in synaptic and epithelial cell junctions. To clarify the biological function of RA175, we disrupted the mouse Igsf4a (Ra175/Tslc1/SynCam/Igsf4a Ra175) gene. Male mice lacking both alleles of Ra175 (Ra175-/-) were infertile and showed oligo-astheno-teratozoospermia; almost no mature motile spermatozoa were found in the epididymis. Heterozygous males and females and homozygous null females were fertile and had no overt developmental defects. RA175 was mainly expressed on the cell junction of spermatocytes, elongating and elongated spermatids (steps 9 to 15) in wild-type testes; the RA175 expression was restricted to the distal site (tail side) but not to the proximal site (head side) in elongated spermatids. In Ra175-/- testes, elongated and mature spermatids (steps 13 to 16) were almost undetectable; round spermatids were morphologically normal, but elongating spermatids (steps 9 to 12) failed to mature further and to translocate to the adluminal surface. The remaining elongating spermatids at improper positions were finally phagocytosed by Sertoli cells. Furthermore, undifferentiated and abnormal spermatids exfoliated into the tubular lumen from adluminal surfaces. Thus, RA175-based cell junction is necessary for retaining elongating spermatids in the invagination of Sertoli cells for their maturation and translocation to the adluminal surface for timely release.     

Cytology of the human seminiferous epithelium

The appearances in cytologic specimens of the principal cell types in the normal human seminiferous epithelium are described and illustrated. Sertoli cells, which are larger than spermatogenic cells, are characterized by a slightly basophilic, ill-defined cytoplasm of triangular, elongated or columnar shape; the cytoplasm may be vacuolated and may contain spermatozoa. The nuclei of Sertoli cells are round, with a uniformly finely granulated chromatin and a single nucleolus. Spermatogenic cells are round or oval and show scanty cytoplasm with deeper basophilia and well-defined cytoplasmic borders. Multinucleation is common in spermatogenic cells. The Sertoli cells constitute a very homogeneous cell population as compared to the spermatogenic cells, which show several distinct cell types (spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa) whose nuclear structures depend on the stage of meiosis. Both cell types may occur as naked nuclei. Some problems of cell classification are discussed.     

Electron microscopic study of spermiogenesis in the free-living marine nematode <Emphasis Type="Italic">Leptosomatides marinae</Emphasis> Platonova 1976 (Enoplida: Leptosomatidae)

The ultrastructure of spermatocytes, spermatids and spermatozoa of the free-living marine nematode Leptosomatides marinae was studied by transmission electron microscopy. In early spermatids, the number of mitochondria, cisterns of the rough endoplasmic reticulum (RER) and dictyosomes increased; the number of membranous organelles (MOs) was insignificant. Later, dictyosomes and MOs filled the cytoplasm. The cytoplasm became distinctly segregated in late spermatids and the MOs concentrated around the nucleus; the mitochondria and organelles of synthesis settled on the cell periphery. Later, a densely packed conglomerate was formed from the central nucleus and a mass of MOs surrounded by an extensive zone of the cytoplasm containing mitochondria and organelles of synthesis. Early spermatozoa had an elongated nucleus surrounded by a layer of cytoplasm containing mitochondria, polarized MOs and bundles of filamentous material, which can be interpreted as fibrous bodies (FBs). The formed spermatozoa had elongated nuclei surrounded by a transparent halo; the cell periphery was a dense matrix, in which MOs and sparse mitochondria were submerged; no FBs were revealed in that phase. In general, the spermatozoa of L. marinae have the main attributes common to the Enoplida spermatozoa, availability of nuclear environment and development of the specific organelles, MOs and FBs, which are not united in complexes.     

Extracellular matrix: recent advances on its role in junction dynamics in the seminiferous epithelium during spermatogenesis

Spermatogenesis takes place in the seminiferous epithelium of the mammalian testis in which one type A1 spermatogonium (diploid, 2n) gives rise to 256 spermatids (haploid, 1n). To accomplish this, developing germ cells, such as preleptotene and leptotene spermatocytes, residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) entering into the adluminal compartment for further development into round, elongating, and elongate spermatids. Recent studies have shown that the basement membrane in the testis (a modified form of extracellular matrix, ECM) is important to the event of germ cell movement across the BTB because proteins in the ECM were shown to regulate BTB dynamics via the interactions between collagens, proteases, and protease inhibitors, possibly under the regulation of cytokines. While these findings are intriguing, they are not entirely unexpected. For one, the basement membrane in the testis is intimately associated with the BTB, which represents the basolateral region of Sertoli cells. Also, Sertoli cell tight junctions (TJs) that constitute the BTB are present side-by-side with cell-cell actin-based adherens junctions (AJ, such as basal ectoplasmic specialization [ES]) and intermediate filament-based desmosome-like junctions. As such, the relative morphological layout between TJs, AJs, and desmosome-like junctions in the seminiferous epithelium is in sharp contrast to other epithelia where TJs are located at the apical portion of an epithelium or endothelium, furthest away from ECM, to be followed by AJs and desmosomes, which in turn constitute the junctional complex. For another, anchoring junctions between a cell epithelium and ECM found in multiple tissues, also known as focal contacts (or focal adhesion complex, FAC, an actin-based cell-matrix anchoring junction type), are the most efficient junction type that permits rapid junction restructuring to accommodate cell movement. It is therefore physiologically plausible, and perhaps essential, that the testis is using some components of the focal contacts to regulate rapid restructuring of AJs between Sertoli and germ cells when germ cells traverse the seminiferous epithelium. Indeed, recent findings have shown that the apical ES, a testis-specific AJ type in the seminiferous epithelium, is equipped with proteins of FAC to regulate its restructuring. In this review, we provide a timely update on this exciting yet rapidly developing field regarding how the homeostasis of basement membrane in the tunica propria regulates BTB dynamics and spermatogenesis in the testis, as well as a critical review on the molecular architecture and the regulation of ES in the seminiferous epithelium.     

Determination of daily sperm production in stallion testes by enumeration of germ cells in homogenates

Thirty adult stallion testes were selected with high (n = 15) and low (n = 15) Daily Sperm Production (DSP)/testis. Parenchymal samples were prepared for morphometric analysis, and the numbers of germ cells and Sertoli cells were determined. Testicular samples were homogenized, and germ cells and Sertoli cells were enumerated using phase contrast microscopy. Numbers of germ cells and Sertoli cells and potential DSP during spermatogenesis were determined. Significant correlations existed between morphometric and homogenate determinations of number per testis of preleptotene, leptotene plus zygotene primary spermatocytes (r = 0.58; P < 0.001), pachytene plus diplotene primary spermatocytes (r = 0.67; P < 0.0001), all primary spermatocytes (r = 0.67; P < 0.0001), round spermatids (r = 0.72; P < 0.0001), and Sertoli cells (r = 0.70; P < 0.0001). Significant correlations (P < 0.0001) existed between morphometric and homogenate determination of DSP/testis based on preleptotene, leptotene plus zygotene primary spermatocytes (r = 0.78), pachytene plus diplotene primary spermatocytes (r = 0.88), and round spermatids (r = 0.85). Using morphometric determination as the standard, the sensitivity (i.e., ability to detect low DSP/testis) and specificity (i.e., ability to detect high DSP/testis) by homogenate enumeration of germ cells was 81 and 93% for round spermatids, 100 and 24% for pachytene plus diplotene primary spermatocytes, and 67 and 87% for preleptotene, leptotene plus zygotene primary spermatocytes, respectively. Enumeration of primary spermatocytes in homogenates was less accurate than enumeration of round or elongated spermatids. Enumeration of round and elongated spermatids in homogenates was a rapid and useful method for determining DSP in horses, and it may prove to be a useful technique for quantitating potential DSP from testicular biopsies.     

Expression of claudin-1 and -11 in immature and mature pheasant (Phasianus colchicus) testes

The expression of claudin-1 and -11, tight junctions (TJs) proteins was examined in immature and adult pheasant (Phasianus colchicus) testes. Claudin-1 and -11 cDNA were highly similar to those of human, mice, and chicken. Claudin-1 mRNA and protein (21 kDa) levels in immature testes were higher than those of adult testis. In immature testes until 6 weeks of age, Claudin-1 was found at contacts between adjacent Sertoli cells and between Sertoli cells and germ cells. In adult testis, Claudin-1 was found in early spermatocytes migrating the blood testis barrier (BTB). Blood vessels were positive for claudin-1. Claudin-11 mRNA and protein (21 kDa) increased during adulthood development of testis. In immature testis, Claudin-11 was found in apicolateral contacts between adjacent Sertoli cells, indicating its involvement in cell adhesion in immature testis. In adult testis, strong wavy Claudin-11 immunoreactivity was parallel to basal lamina at the basal part of seminiferous epithelium, indicating that Claudin-11 at the inter-Sertoli TJs may act as a structural element of the BTB. Weak Claudin-1 and -11 immunoreactivity at contacts between Sertoli cells to elongating/elongated spermatids, meiotic germ cells, and basal lamina suggests that they also participate in the cell-cell and cell-extracellular matrix adhesion in pheasant testis. Testosterone increased claudin-11 mRNA in testis organ culture and Sertoli cell primary culture, suggesting positive regulation of claudin-11 gene by androgen in Sertoli cells of pheasant testis. This is the first report on the claudins expression at BTB in avian testis.     

Actin-related intercellular adhesion junctions in the germinal compartment of the testis in the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus)

The germinal epithelium of male vertebrates consists of Sertoli cells and spermatogenic cells. Intercellular junctions formed by Sertoli cells assume critical roles in the normal functions of this epithelium. While Sertoli cell junctions have been well characterized in mammals, similar junctions in nonmammalian vertebrates have received little attention. We examined the intercellular junctions found within the germinal epithelium of the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus). Ultrastructurally, Sertoli cells were seen to form filament-associated junctions in both species. Adjacent Sertoli cells formed microfilament-related junctions near their apices. Filaments of these junctions were arranged in loose networks and were not associated with cisterns of endoplasmic reticulum. In fixed, frozen sections of hagfish testis, similar areas labeled with rhodamine phalloidin, indicating the filament type is actin. In the lamprey, desmosomes were observed immediately below the microfilament-related junctions. In appearance and location, the Sertoli cell junctions observed in these species resembled those of the typical junctional complex of other epithelial cell types. No junctions were observed between Sertoli cells and elongating spermatids. In the hagfish, but not the lamprey, an additional zone of microfilaments occurred near the base of Sertoli cells in areas of association with the basal lamina. Our observations are consistent with the proposal that the unique forms of intercellular attachment found in the testes of higher vertebrates evolved from a typical epithelial form of intercellular junction.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli-Sertoli and Sertoli-germ cell interface

The coxsackie and adenovirus receptor (CAR), a putative cell-cell adhesion molecule, has attracted wide interest due to its importance in viral pathogenesis and in mediating adenoviral gene delivery. However, the distribution pattern and physiological function of CAR in the testis is still not clear. Here, we identified CAR in Sertoli cells and germ cells of rats. In vivo studies have shown that CAR resides at the blood-testis barrier as well as at the ectoplasmic specialization. The persistent expression of CAR in rat testes from neonatal period throughout adulthood implicates its role in spermatogenesis. Using primary Sertoli cell cultures, we observed a significant induction of CAR during the formation of Sertoli cell epithelium. Furthermore, CAR was seen to be concentrated at inter-Sertoli cell junctions, co-localizing with tight junction protein marker ZO-1 and adherens junction protein N-cadherin. CAR was also found to be associated with proteins of Src kinase family and its protein level declined after TNFα treatment in Sertoli cell cultures. Immunofluorescent staining of isolated germ cells has revealed the presence of CAR on spermatogonia, spermatocytes, round spermatids and elongate spermatids. Taken together, we propose that CAR functions as an adhesion molecule in maintaining the inter-Sertoli cell junctions at the basal compartment of the seminiferous epithelium. In addition, CAR may confer adhesion between Sertoli and germ cells at the Sertoli-germ cell interface. It is possible that the receptor utilized by viral pathogens to breakthrough the epithelial barrier was also employed by developing germ cells to migrate through the inter-Sertoli cell junctions.     

Effect of an acute exposure of rat testes to gamma rays on germ cells and on Sertoli and Leydig cell functions.

Germ cells and Sertoli and Leydig cell functions were studied from 7 to 180 days after an acute exposure of 2-month-old rat testes to 9 Gy of gamma rays. Body weight, testis and epididymal weights were recorded. Sertoli cell parameters (androgen-binding protein, ABP, in caput epididymis and plasma follicle stimulating hormone, FSH) and Leydig cell parameters (plasma luteinizing hormone, LH, testosterone and prostate and seminal vesicle weights) were determined together with the number of germ cells and Sertoli cells. Irradiation did not affect body weight but significantly reduced testicular and epididymal weights from day 7 and day 15 post-irradiation respectively. The cells killed by irradiation were mainly spermatogonia and preleptotene spermatocytes engaged in replicating their DNA at the time of exposure, but all spermatocytes seemed damaged as they gave abnormal descendent cells. By day 34, only elongated spermatids remained in a few tubules and thereafter very little regeneration of the seminiferous epithelium occurred, except for one rat which showed a better regeneration. Levels of ABP decreased by day 15 when the germ cell depletion had reached the pachytene spermatocytes, whereas FSH and LH levels rose when the number of elongated spermatids decreased. Levels of testosterone and the weight of the seminal vesicles did not change; occasionally, the prostate weight was slightly reduced. These results support our hypothesis that pachytene spermatocytes and elongated spermatids are involved in influencing some aspects of Sertoli cell function in the adult rat.     

Improvement of spermatogenesis in adult cryptorchid rat testis by intratesticular infusion of lactate.

In order to test the hypothesis that a lack of energy could be a cause of germ cell death at high temperatures, cryptorchid rats testes were infused with lactate, delivered by osmotic pumps over 3-15 days. In cryptorchid testes, the spermatids and spermatocytes were lost between 3 and 8 days. In cryptorchid testes supplemented with lactate, elongated spermatids persisted in a few seminiferous tubules at Day 15. Elimination of round spermatids occurred progressively between 3 and 15 days, mostly at stage VIII. The loss of spermatocytes increased after 8 days, and 30% of seminiferous tubules still contained meiotic or meiotic plus spermiogenetic cells at Day 15. After 8 days, the chromatin of step 8 round spermatids was abnormal and nuclear elongation did not commence. The Sertoli cell cytoplasm that was retracted toward the basal compartment of the seminiferous epithelium could not hold the germ cells of the adluminal compartment. Therefore, attachment of germ cells to Sertoli cells and the supply of lactate seem necessary for the development of germ cells at high temperatures. The improvement in spermatogenesis in cryptorchid supplemented testes for several days is a new finding.     

Actin-related intercellular junctions in the germinal compartment of the testis in Poecilia reticulata (Teleostei,Poeciliidae)

Summary In this paper we present evidence for the presence of actin-related junctions between neighboring Sertoli cells and between Sertoli cells and spermatids in the testis of the guppy (Poecilia reticulata). In the guppy, spermatogenesis occurs in spermatocysts that are lined by a simple squamous to cuboidal epithelium formed of Sertoli cells. At a certain stage of differentiation, elongate spermatids occur in Sertoli cell recesses in the apical surface of Sertoli cells. When evaluated by electron microscopy, junctions occur between Sertoli cells and spermatids situated in the recesses. In these regions, obvious linkages occur between the plasma membrane of Sertoli cell recesses and the adjacent spermatids. Moreover, large concentrations of microfilaments occur in the Sertoli cell cytoplasm immediately underlying the crypts. Also, junctional complexes are apparent between neighboring Sertoli cells near the apical surface of the epithelium. These complexes consist of microfilament-related components (probably contributing to both tight and adhesion junctions), which occur closest to the lumen, and intermediate-filament related desmosomes, which occur more basally. In fixed frozen sections of guppy testis, probes for filamentous actin (rhodamine phalloidin) and myosin II (polyclonal antisera raised against human platelet myosin II) react with function regions between neighboring Sertoli cells and between Sertoli cells and spermatids. We conclude that actin-related junctions occur at both these sites and that the actin networks have contractile properties because they contain myosin II.     

Testicular involution following optic enucleation

Summary The testes of adult male Syrian hamsters underwent involution within six weeks after optic enucleation. The diameter of the seminiferous tubules was 39% less than controls. Sertoli cells, spermatogonia, and primary spermatocytes were still present, but all steps of spermatids were completely absent from the involuted testes. Lipid droplets filled the Sertoli cell cytoplasm and often encroached upon the nucleus. Sertoli cells had sparse mitochondria and smooth endoplasmic reticulum, but Golgi cisternae were abundant. Typical SertoliSertoli junctions attached contiguous Sertoli cells. With lanthanum tracers it was demonstrated that these junctions were impenetrable; therefore, the bloodtestis barrier was deemed intact. Irregularly shaped protrusions often arose from the peritubular tissue and extended inward toward the seminiferous epithelium, often displacing the cytoplasm of the Sertoli cells and spermatogonia. The core of these protrusions consisted of irregular extensions of myoid cell cytoplasm surrounded by the myoid cells' basal lamina. External to the myoid cell basal lamina were bundles of collagen filaments with the basal lamina of the seminiferous epithelium forming the outermost layer of these protrusions. The apices of the Sertoli cells gave rise to numerous leaf-like processes that extended into and obliterated the lumen of the tubules. The Sertoli cell basal cytoplasm often contained phagocytized degenerating germ cells that appeared to give rise to the lipid droplets that filled the Sertoli cell cytoplasm. Acid phosphatase rich lysosome-like organelles were seen fusing with the degenerating germ cells and lipid droplets. The degenerating germ cells also were shown to contain acid phosphatase activity.     

The fine structural localization of testicular phosphatases in man: The control testis

Summary Electron microscopic cytochemistry was used to determine the localization of five phosphatase enzymes—glucose-6-phosphatase, inosine diphosphatase, thiamine pyrophosphatase, acid phosphatase, and adenosine triphosphatase—in control human testes. Glucose-6-phosphatase occurred in the endoplasmic reticulum and nuclear envelope of Sertoli cells, Leydig cells and primitive spermatogonia, but was not observed in more advanced spermatogenic cells. The presence of glucose-6-phosphatase activity paralleled the presence of glycogen in spermatogenic cells, i.e., both occurred in type AL and AD spermatogonia but not in type AP or B spermatogonia or in more advanced spermatogenic cells. Inosine diphosphatase activity was found in the endoplasmic reticulum, nuclear envelope, and Golgi complex of Sertoli cells and all spermatogenic cells except late spermatids. Additionally, inosine diphosphatase activity was localized at the junctions between Sertoli cells and late spermatids, but was not associated with any other plasma membrane. Thiamine pyrophosphatase reaction product was found in the Golgi bodies of Sertoli cells and in spermatogenic cells through immature spermatids. Neither inosine diphosphatase nor thiamine pyrophosphatase was observed in the Golgi bodies of spermatids during acrosomal formation. Acid phosphatase activity was found in lysosomes of spermatogonia, spermatocytes, and spermatids, in lysosomes of Leydig cells, and in lysosomes, lipofuscin bodies, and Golgi cisternae of Sertoli cells. It is thought that Sertoli lysosomes play a role in the phagocytosis of degenerating germ cells; however, the role of spermatogenic or Leydig lysosomes is unknown. Adenosine triphosphatase activity occurred at the interfaces between two spermatogonia, and between Sertoli cells and spermatogonia, but was not observed in the spaces between two Sertoli cells, two spermatocytes, two spermatids, or between Sertoli cells and spermatocytes, or between Sertoli cells and spermatids.Supported in part by a grant from the U.S. Atomic Energy Commission (AT-(40-1)-4002).     

Ectoplasmic specialization: a friend or a foe of spermatogenesis?

The ectoplasmic specialization (ES) is a testis-specific, actin-based hybrid anchoring and tight junction. It is confined to the interface between Sertoli cells at the blood-testis barrier, known as the basal ES, as well as between Sertoli cells and developing spermatids designated the apical ES. The ES shares features of adherens junctions, tight junctions and focal contacts. By adopting the best features of each junction type, this hybrid nature of ES facilitates the extensive junction-restructuring events in the seminiferous epithelium during spermatogenesis. For instance, the alpha6beta1-integrin-laminin 333 complex, which is usually limited to the cell-matrix interface in other epithelia to facilitate cell movement, is a putative apical ES constituent. Furthermore, JAM-C and CAR, two tight junction integral membrane proteins, are also components of apical ES involving in spermatid orientation. We discuss herein the mechanisms that maintain the cross-talk between ES and blood-testis barrier to facilitate cell movement and orientation in the seminiferous epithelium.     

Cell to cell relationships in the seminiferous epithelium in the mouse embryo

Summary Germ cells and Sertoli cells in embryonic mouse testes (day 14 to 20 of gestation) were examined by sectioning and freeze-fracture. Intercellular cytoplasmic bridges between the germ cells are observed in day 14 and older embryos. Membrane specializations with dense fuzzy material similar to the socalled desmosome-like structures are found between Sertoli cells and germ cells. A cell contact area with dense opposed membranes is also found between adjacent germ cells. Asymmetrical dense fuzzy lining of both Sertoli and germ cell membranes is noted. Pinocytotic pits or caveolae are frequently found in the Sertoli cell membrane. Between adjacent Sertoli cells, gap junctions of various sizes and focal meshworks of the occluding junctions are found. Most of the occluding junctional particles are located in the center of the grooves in the E face, and are similar to those in postnatal and adult Sertoli cell junctions. In addition, on both fractured faces there are ridges and grooves devoid of particles which are continuous with occluding junctions with particles, suggesting an initial stage in the formation of occluding junctions of the Sertoli cells. Particles gathered at the site of desmosome-like structures are present on the P face of the Sertoli cell.This work is supported by the Japanese Ministry of Education     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Differentiation of Primary Spermatocytes to Elongated Spermatids by Mammalian FSH in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster

Our previous studies (10, 11) showed that mammalian follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the initiation and promotion of spermatogenesis from secondary spermatogonia to primary spermatocytes in organ culture of testes fragments from the newt, Cynops pyrrhogaster. The present study demonstrated that FSH promoted in the same model system the differentiation of primary spermatocytes even further: to the stage of elongated spermatids. When testes fragments, consisting of somatic cells and germ cells (mostly primary spermatocytes), were cultured in a control medium for three weeks, only round spermatids and spermatogonia were observed; both the diameter of the cysts and the viability of the germ cells decreased to about 10–15% of the original level. On the other hand, when the medium was supplemented with FSH, elongated spermatids appeared by the second week; both the diameter of the cysts and the viability of the germ cells were maintained at a higher level than in the control medium. The effect of FSH was dose-dependent. However, neither transferrin, androgens (testosterone and 5α-dihydrotestosterone) nor luteinizing hormone (LH) was effective. The addition of cyanoketone, a specific inhibitor of 3β-hydroxy-Δ⁵-steroid dehydrogenase (3β-HSD) (32), to the FSH-containing medium did not prevent the differentiation promoted by FSH, indicating that it is unlikely that Δ⁴-steroid metabolites produced in fragments by FSH acted directly on germ cells. Insulin was found to improve the viability of germ cells during a 2 week of culture period. In the presence of FSH, the cells in various differentiative stages had morphological characteristics very similar to those in vivo, whereas in the absence of FSH primary spermatocytes showed abnormal features in their nuclei and cytoplasm, indicating that they were deteriorating. These results and our previous results (1–3) suggest that FSH promotes primary spermatocytes to differentiate into elongated spermatids probably by stimulating Sertoli cells to secrete factors which then act on the germ cells.