Kit ligand mediates survival of type a spermatogonia and dividing spermatocytes in postnatal mouse testes

In the mouse testis, spontaneous death of spermatogonia has a large impact on the output of differentiating spermatids. The tyrosine kinase receptor c-kit is expressed in type A, intermediate, and B spermatogonia, and kit-ligand (KL) is expressed in Sertoli cells. Previous work indicated a depletion of type A spermatogonia after in vivo exposure to an antibody that blocks c-kit function. The present work was undertaken to determine whether blocking c-kit function results in apoptosis of spermatogonia or in an inability of spermatogonia to proliferate. Testes sections were stained by a method that detects apoptotic cells in situ. In testes of 8-day postnatal (P8) males, type A spermatogonia are the predominant germ cell type present. Stained sections from P8 males injected with the c-kit antagonistic antibody ACK2 showed a fivefold higher rate of cell death than uninjected controls. At least a twofold increase was observed in P12 and P30 injected males and in P30 SI^d + males as compared to uninjected controls. Determination of the stage of germ cell development that was affected in P30 males indicated that the frequency of gonial cell death was increased fourfold, but the frequency of death in spermatocytes around the time of the meiotic division was increased 15-fold. It is concluded that KL acts to prevent apoptosis in the testis in vivo, that the membrane bound form of KL may be more effective, and that survival of late meiotic and dividing spermatocytes is regulated by KL through an indirect mechanism probably mediated by Sertoli cells. Thus, KL is an important regulator of spermatid output. © 1995 wiley-Liss, Inc.     

Immunolocalization of proliferating cell nuclear antigen (PCNA) in cynomolgus monkey (Macaca fascicularis) testes during postnatal development

The age-related distribution of proliferating cell nuclear antigen (PCNA) in the testes of cynomolgus monkeys (Macaca fascicularis) during postnatal development was detected using light-microscopic immunohistochemistry. In neonatal testes, some PCNA-positive spermatogonia, Sertoli cells, peritubular cells, and Leydig cells were detected. In early infantile testes, only a few of these cell types were positive. In late infantile testes, the numbers of positive cells were greater than in the earlier developmental stages. In pubertal testes, the numbers of positive spermatogonia, spermatocytes, Sertoli cells, peritubular cells, and Leydig cells were considerably higher. In adult testes, a larger percentage of spermatogonia and spermatocytes was positive, and peritubular cells and Leydig cells were occasionally positive; secondary spermatocytes, spermatids, and Sertoli cells were not positive. We concluded that immunolocalization of PCNA can serve as a tool for studying proliferation status in developing testes of cynomolgus monkeys. A relatively low proliferative activity in early infantile testes and a remarkable increase of proliferative activity in pubertal testes correlate with the fluctuations of steroidogenic functions during postnatal development in cynomolgus monkeys.     

Gene expression alterations by conditional knockout of androgen receptor in adult Sertoli cells of Utp14b jsd/jsd (jsd) mice

Spermatogenesis is dependent primarily on testosterone action on the Sertoli cells, but the molecular mechanisms have not been identified. Attempts to identify testosterone-regulated target genes in Sertoli cells have used microarray analysis of gene expression in mice lacking the androgen receptor (AR) in Sertoli cells (SCARKO) and wild-type mice, but the analyses have been complicated both by alteration of germ cell composition of the testis when pubertal or adult mice were used and by differences in Sertoli-cell gene expression from the expression in adults when prepubertal mice were used. To overcome these limitations and identify AR-regulated genes in adult Sertoli cells, we compared gene expression in adult jsd (Utp14b jsd/jsd, juvenile spermatogonial depletion) mouse testes and with that in SCARKO-jsd mouse testes, since their cellular compositions are essentially identical, consisting of only type A spermatogonia and somatic cells. Microarray analysis identified 157 genes as downregulated and 197 genes as upregulated in the SCARKO-jsd mice compared to jsd mice. Some of the AR-regulated genes identified in the previous studies, including Rhox5, Drd4, and Fhod3, were also AR regulated in the jsd testes, but others, such as proteases and components of junctional complexes, were not AR regulated in our model. Surprisingly, a set of germ cell–specific genes preferentially expressed in differentiated spermatogonia and meiotic cells, including Meig1, Sycp3, and Ddx4, were all upregulated about 2-fold in SCARKO-jsd testes. AR-regulated genes in Sertoli cells must therefore be involved in the regulation of spermatogonial differentiation, although there was no significant differentiation to spermatocytes in SCARKO-jsd mice. Further gene ontogeny analysis revealed sets of genes whose changes in expression may be involved in the dislocation of Sertoli cell nuclei in SCARKO-jsd testes.     

Effect of the W mutation, for white belly spot, on testicular germ cell differentiation in mice.

The effect of the mutation for white belly spot controlled by the dominant gene W on spermatogenesis in mice was examined by experimental cryptorchidism and its surgical reversal. The course of spermatogenesis from spermatogonia to spermatid was normal in intact testes of W/+ mice. In cryptorchid testes, there was no difference in the number and activity of Type A spermatogonia between the testes of W/+ and +/+ mice, in mitotic and labelling indices. Although surgical reversal of the cryptorchid testis resulted in regenerative differentiation of germ cells in both genotypes, the recovery of cell differentiation in the W/+ testis was slower than in the +/+ testis. There were fewer germ cells, such as intermediate-Type B spermatogonia or more advanced ones, in W/+ testes. On Day 17 after surgical reversal, cell associations in W/+ testes were abnormal and the numbers of intermediate-Type B spermatogonia, spermatocytes and spermatids were approximately 70, 50 and 15%, respectively, of those in +/+ testes. These results indicate that the W gene affects spermatogenic cell differentiation in adult mice.     

Developmental expression of the translocator protein 18 kDa (TSPO) in testicular germ cells

Translocator protein (TSPO) is a high affinity 18 kDa drug- and cholesterol-binding protein strongly expressed in steroidogenic tissues where it mediates cholesterol transport into mitochondria and steroid formation. Testosterone formation by Leydig cells in the testis is critical for the regulation of spermatogenesis and male fertility. Male germ cell development comprises two main phases, the pre-spermatogenesis phase occurring from fetal life to infancy and leading to spermatogonial stem cell (SSC) formation, and spermatogenesis, which consists of repetitive cycles of germ cell mitosis, meiosis and differentiation, starting with SSC differentiation and ending with spermiogenesis and spermatozoa formation. Little is known about the molecular mechanisms controlling the progression from one germ cell phenotype to the next. Here, we report that testicular germ cells express TSPO from neonatal to adult phases, although at lower levels than Leydig cells. TSPO mRNA and protein were found at specific steps of germ cell development. In fetal and neonatal gonocytes, the precursors of SSCs, TSPO appears to be mainly nuclear. In the prepubertal testis, TSPO is present in pachytene spermatocytes and dividing spermatogonia. In adult testes, it is found in a stage-dependent manner in pachytene spermatocyte and round spermatid nuclei, and in mitotic spermatogonia. In search of TSPO function, the TSPO drug ligand PK 11195 was added to isolated gonocytes with or without the proliferative factors PDGF and 17β-estradiol, and was found to have no effect on gonocyte proliferation. However, TSPO strong expression in dividing spermatogonia suggests that it might play a role in spermatogonial mitosis. Taken together, these results suggest that TSPO plays a role in specific phases of germ cell development.     

Cloning and characterization of the gene encoding the bovine BOULE protein

The Deleted in Azoospermia (DAZ) genes encode potential RNA-binding proteins that are expressed exclusively in the germ-line. The bovine Deleted in Azoospermia-like gene is a strong candidate for male cattle-yak infertility. In this work, with the goe goal to further reveal the genetic cause of male cattle-yak sterility, another bovine DAZ family gene, b-boule, was isolated and characterized. The b-boule gene is predicted to encode a polypeptide of 295 amino acids with an RNP-type RNA recognition domain. Tertiary structure analysis shows that b-boule binds specifically to polypyrimidine RNAs and might act as a nuclear ribonucleoprotein particle auxiliary factor during germ cell formation and morphological changes of germ cells. RT-PCR assays revealed that b-boule was expressed specifically in the adult testis. However, an extremely low level of expression was detected in the testis of sterile male cattle-yaks. Microstructure of the testes from sterile males showed that type A spermatogonia were the only germ cells present and that few germ cells developed further than the stage of pachytene spermatocytes. These results suggest that b-boule may function in bovine spermatogenesis, and that low levels of b-boule expression might lead to male sterility in cattle-yaks.     

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.     

Murine polo like kinase 1 gene is expressed in meiotic testicular germ cells and oocytes

To identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding region shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythroleukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the PlK1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage. © 1995 Wiley-Liss, Inc.     

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.     

Morphological and histochemical characterization of the seminiferous epithelial and Leydig cells of the turkey

Unlike mammals, there is little fundamental information about spermatogenesis in birds. This study was undertaken to clarify the morphology, histochemistry, and lectin affinity of the seminiferous epithelial cells and Leydig cells in pre-pubertal (8- to 15-week old) and adult (40- to 44-week old) domestic turkeys. In adult turkeys, three types of spermatogonia were defined based on their chromatin distribution and nuclear morphology: the dark type A (A(d)); the pale type A (A(p)); and the type B. The A(d) is the least numerous and least conspicuous and consequently difficult to locate. Based on its spatial distribution and overall morphology, type A(d) spermatogonia were postulated to be the spermatogonia stem cells in the turkey. Antibodies to c-kit were localized to spermatogonia in the pre-pubertal and to a lesser extent in adult males. Peanut agglutinin (PNA) was specific for spermatocytes in the pre-pubertal males and spermatogonia and early spermatocytes in adult males. Wheat-germ agglutinin (WGA) highlighted Sertoli cells in both age groups. Bandeiraea simplicifolia I, soybean agglutinin, and winged-pea agglutinin staining were limited to the wall of the seminiferous tubule and some extra-tubular cell types. Concanavalin A staining was diffuse and not cell-specific and, therefore, could not be used to selectively identify a particular cell type. It was concluded that WGA and PNA could aid in identifying specific cell types in the seminiferous epithelium of testis from pre-pubertal and mature turkeys. Only Leydig cells were alkaline phosphatase reactive in the mature turkey testes. The information from this study is being used to adapt techniques for the isolation and partial purification developed for mammalian spermatogonia to avian spermatogonia and other specific cell types in the testes.     

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.     

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.     

Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis

Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/Wv mutant mouse testis. In the present study, some of the W/Wv mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/Wv mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/Wv spermatogonia into the seminiferous tubules of normal SCF (W/Wv) or SCF mutant (Sl/Sld) mice demonstrated that the W/Wv spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/Wv testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/Wv testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.     

Reduced sperm count and normal fertility in male mice with targeted disruption of the ADP-ribosylation factor-like 4 (Arl4) gene

The ADP-ribosylation factor-like protein 4 (ARL4) is a 22-kDa GTP-binding protein which is abundant in testes of pubertal and adult rodents but absent in testes from prepubertal animals. During testis development, ARL4 expression starts at day 16 when the spermatogenesis proceeds to the late pachytene. In the adult testis, the ARL4 protein was detected in pre- and postmeiotic cells, spermatocytes, and spermatides, but not in spermatogonia and mature spermatozoa. Mouse Arl4-null mutants generated by targeted disruption of the Arl4 gene were viable and grew normally; male as well as female Arl4(-/-) mice were fertile. However, inactivation of the Arl4 gene resulted in a significant reduction of testis weight and sperm count by 30 and 60%, respectively, without reduction of litter size or frequency. It is suggested that the disruption of Arl4 produces a moderate retardation of germ cell development, possibly at the stage of meiosis.     

The Transcriptional Cell Atlas of Testis Development in Sheep at Pre-Sexual Maturity

Sheep testes undergo a dramatic rate of development with structural changes during pre-sexual maturity, including the proliferation and maturation of somatic niche cells and the initiation of spermatogenesis. To explore this complex process, 12,843 testicular cells from three males at pre-sexual maturity (three-month-old) were sequenced using the 10× Genomics Chromium^TM single-cell RNA-seq (scRNA-seq) technology. Nine testicular somatic cell types (Sertoli cells, myoid cells, monocytes, macrophages, Leydig cells, dendritic cells, endothelial cells, smooth muscle cells, and leukocytes) and an unknown cell cluster were observed. In particular, five male germ cell types (including two types of undifferentiated spermatogonia (A_pale and A_dark), primary spermatocytes, secondary spermatocytes, and sperm cells) were identified. Interestingly, A_pale and A_dark were found to be two distinct states of undifferentiated spermatogonia. Further analysis identified specific marker genes, including UCHL1, DDX4, SOHLH1, KITLG, and PCNA, in the germ cells at different states of differentiation. The study revealed significant changes in germline stem cells at pre-sexual maturation, paving the way to explore the candidate factors and pathways for the regulation of germ and somatic cells, and to provide us with opportunities for the establishment of livestock stem cell breeding programs.     

Drosophila RecQ5 is involved in proper progression of early spermatogenesis

RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.