Loss of TSLC1 causes male infertility due to a defect at the spermatid stage of spermatogenesis

Tumor suppressor of lung cancer 1 (TSLC1), also known as SgIGSF, IGSF4, and SynCAM, is strongly expressed in spermatogenic cells undergoing the early and late phases of spermatogenesis (spermatogonia to zygotene spermatocytes and elongating spermatids to spermiation). Using embryonic stem cell technology to generate a null mutation of Tslc1 in mice, we found that Tslc1 null male mice were infertile. Tslc1 null adult testes showed that spermatogenesis had arrested at the spermatid stage, with degenerating and apoptotic spermatids sloughing off into the lumen. In adult mice, Tslc1 null round spermatids showed evidence of normal differentiation (an acrosomal cap and F-actin polarization indistinguishable from that of wild-type spermatids); however, the surviving spermatozoa were immature, malformed, found at very low levels in the epididymis, and rarely motile. Analysis of the first wave of spermatogenesis in Tslc1 null mice showed a delay in maturation by day 22 and degeneration of round spermatids by day 28. Expression profiling of the testes revealed that Tslc1 null mice showed increases in the expression levels of genes involved in apoptosis, adhesion, and the cytoskeleton. Taken together, these data show that Tslc1 is essential for normal spermatogenesis in mice.     

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.     

Indirect Sertoli cell-mediated ablation of germ cells in mice expressing the inhibin-alpha promoter/herpes simplex virus thymidine kinase transgene

In the present study, we describe a novel mouse model for inducible germ cell ablation. The mice express herpes simplex virus thymidine kinase (HSV-TK) under the inhibin-alpha subunit promoter (Inhalpha). When adult transgenic (TG) mice were treated with famciclovir (FCV) for 4 wk, their spermatogenesis was totally abolished, with only Sertoli cells and few spermatids remaining in the seminiferous tubules. However, testicular steroidogenesis was not affected. Shorter treatment periods allowed us to follow up the progression of germ cell death: After 3 days, spermatogonia and preleptotene spermatocytes were no longer present. After a 1-wk treatment, spermatogonia, preleptotene, and zygotene spermatocytes were missing and the amount of pachytene spermatocytes was decreased. After a 2-wk treatment, round and elongating spermatids were present. During the third week, round spermatids were lost and, finally, after a 4-wk treatment, only Sertoli cells and few spermatids were present. Interestingly, the transgene is detected in Leydig and Sertoli cells but not in spermatogonia. This suggests that FCV is phosphorylated in Sertoli cells, and thereafter, leaks to neighboring spermatogonia, apparently through cell-cell junctions present, enabling trafficking of phosphorylated FCV. Because of the many mitotic divisions they pass through, the spermatogonia are very sensitive to toxins interfering with DNA replication, while nondividing Sertoli cells are protected. Using transillumination-assisted microdissection of the seminiferous tubules, the gene-expression patterns analyzed corresponded closely to the histologically observed progression of cell death. Thus, the model offers a new tool for studies on germ cell-Sertoli cell interactions by accurate alteration of the germ cell composition in seminiferous tubules.     

Excess type I interferon signaling in the mouse seminiferous tubules leads to germ cell loss and sterility

Type I (α and β) interferons (IFNs) elicit antiproliferative and antiviral activities via the surface receptor IFNAR. Serendipitous observations in transgenic mice in 1988 strongly suggested that IFNα/β overexpression in the testis disrupts spermatogenesis. Here, we compare a new mouse strain transgenic for IFNβ (Tg10) and a sister strain lacking the IFNAR1 subunit of IFNAR (Tg10-Ifnar1(-/-)), both strains expressing the transgene in the testis. The main source of IFNβ RNA was the spermatid population. Importantly, the Tg10 mice, but not the double mutant Tg10-Ifnar1(-/-), showed altered spermatogenesis. The first IFNAR-dependent histological alteration was a higher apoptosis index in all germ cell categories apart from non-dividing spermatogonia. This occurred 3 weeks after the onset of IFNβ production at postnatal day 20 and in the absence of somatic cell defects in terms of cell number, expression of specific cell markers, and hormonal activities. Several known interferon-stimulated genes were up-regulated in Tg10 Sertoli cells and prepachytene germ cells but not in pachytene spermatocytes and spermatids. In concordance with this, pachytene spermatocytes and spermatids isolated from wild-type testes did not display measurable amounts of IFNAR1 and phosphorylated STAT1 upon IFNβ challenge in vitro, suggesting hyporesponsiveness of these cell types to IFN. At day 60, Tg10 males were sterile, and Sertoli cells showed increased amounts of anti-Mullerian hormone and decreased production of inhibin B, both probably attributable to the massive germ cell loss. Type I interferon signaling may lead to idiopathic infertilities by affecting the interplay between germ cells and Sertoli cells.     

IGSF4: a new intercellular adhesion molecule that is called by three names,TSLC1, SgIGSF and SynCAM,by virtue of its diverse function

Members of the immunoglobulin superfamily often play key roles in intercellular adhesion. IGSF4 is a novel immunoglobulin (Ig)-like intercellular adhesion molecule. Three Ig-like domains are included in the extracellular domain of IGSF4 and mediate homophilic or heterophilic interactions independently of Ca2+. The cytoplasmic domain of IGSF4 contains the binding motifs that connect to actin fibers. Since IGSF4 has been characterized by several independent research groups, this molecule is called by three names, TSLC1, SgIGSF and SynCAM. IGSF4 was first characterized as a tumor suppressor of non-small cell lung cancer and termed TSLC1, although how IGSF4 suppresses tumor growth remains unknown. Silencing of the IGSF4 gene was primarily achieved by allelic loss and promoter methylation in this type of cancers. Soon after this discovery, IGSF4 was found to have roles in adhesion of spermatogenic cells to Sertoli cells and mast cells to fibroblasts and termed SgIGSF. Other researchers revealed that IGSF4 drives synaptic formation of neural cells and termed it SynCAM.     

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).     

Expression and functional characterization of the adhesion molecule spermatogenic immunoglobulin superfamily in the mouse testis

Spermatogenic immunoglobulin superfamily (SgIGSF) is a mouse protein belonging to the immunoglobulin superfamily expressed in the spermatogenic cells of seminiferous tubules. We produced a specific polyclonal antibody against SgIGSF. Western blot analysis of the testes from postnatal developing mice using this antibody demonstrated multiple immunopositive bands of 80-130 kDa, which increased in number and size with the postnatal age. Enzymatic N-glycolysis caused reduction in the size of these bands to 70 kDa, indicating that SgIGSF is a glycoprotein and its glycosylation pattern and extent are developmentally regulated. Immunohistochemical analysis of the adult testis demonstrated that SgIGSF was present in the spermatogenic cells in the earlier steps of spermatogenesis and increased in amount from intermediate spermatogonia through zygotene spermatocytes but was diminished in the steps from early pachytene spermatocytes through round spermatids. After meiosis, SgIGSF reappeared in step 7 spermatids and was present in the elongating spermatids until spermiation. The immunoreactivity was localized primarily on the cell membrane. Consistent with the findings in adult testes, the analysis of the developing testes revealed that SgIGSF was expressed separately in the spermatogenic cells in earlier and later phases. Sertoli cells had no expression of SgIGSF, whereas both SgIGSF immunoprecipitated from the testis lysate and produced in COS-7 cells was shown to bind to the surface of Sertoli cells in primary culture. These results suggested that SgIGSF on the surface of spermatogenic cells binds to some membrane molecules on Sertoli cells in a heterophilic manner and thereby may play diverse roles in the spermatogenesis.     

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.     

A Biological Model of Accelerated Senescence: II. Age-Related Changes in the Number of Developing Male Germ Cells and Sertoli Cells in the Gonads of Senescence-Accelerated Mice

It was shown that in immature three- to four-week-old mice prone to accelerated senescence (SAMP1 strain), the number of spermatogonia, pachytene spermatocytes, and circular spermatids exceeded that in mice resistant to accelerated senescence (SAMR1 strain) by more than two times. Differences were found in the pattern of age-related changes in the number of meiotic and postmeiotic cells in the sexually mature SAMP1 and SAMR1 mice. In the gonads of SAMP1 and SAMR1 mice, the number of Sertoli cells was unstable.     

IGSF11 is required for pericentric heterochromatin dissociation during meiotic diplotene

Meiosis initiation and progression are regulated by both germ cells and gonadal somatic cells. However, little is known about what genes or proteins connecting somatic and germ cells are required for this regulation. Our results show that deficiency for adhesion molecule IGSF11, which is expressed in both Sertoli cells and germ cells, leads to male infertility in mice. Combining a new meiotic fluorescent reporter system with testicular cell transplantation, we demonstrated that IGSF11 is required in both somatic cells and spermatogenic cells for primary spermatocyte development. In the absence of IGSF11, spermatocytes proceed through pachytene, but the pericentric heterochromatin of nonhomologous chromosomes remains inappropriately clustered from late pachytene onward, resulting in undissolved interchromosomal interactions. Hi-C analysis reveals elevated levels of interchromosomal interactions occurring mostly at the chromosome ends. Collectively, our data elucidates that IGSF11 in somatic cells and germ cells is required for pericentric heterochromatin dissociation during diplotene in mouse primary spermatocytes.     

Dehydrodolichyl diphosphate synthase in Sertoli and spermatogenic cells of prepuberal rats

The specific activity of 2,3-dehydrodolichyl diphosphate synthase in homogenates of protease-treated seminiferous tubules, enriched spermatogenic cells, and Sertoli cells changed as a function of the age of prepuberal rats. The highest enzymatic activity occurred in each case in 23-day-old rats. Homogenates of pachytene spermatocytes, spermatids, or Sertoli cells had higher synthase activity than a whole testicular homogenate prepared by protease treatment of tubules. Enzymatic activity in pachytene spermatocytes expressed per mg of protein was about 1.7-fold higher than in spermatids, 5.3-fold higher than in spermatogonia, and about 8.3-fold higher than in spermatozoa. Therefore, the increase in spermatogenic cell synthase before day 23 can be accounted for by the appearance of the pachytene spermatocytes. Enzymatic activity decreased remarkably after the differentiation of spermatids into spermatozoa. Synthase activity in enriched Sertoli cell preparations was 1.5-2.3-fold higher than in spermatogenic cell preparations between days 15 and 30. Therefore, both spermatogenic cells and Sertoli cells contribute to changes in the enzymatic activity in seminiferous tubules during development. These changes may be important in regulating the availability of dolichyl phosphate for glycoprotein synthesis during early stages of differentiation.     

beta-Nerve growth factor participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes

NGF appears to be involved in spermatogenesis. However, mice lacking NGF or TrkA genes do not survive more than a few days whereas p75(NTR) knockout mice are viable and fertile. Therefore, we addressed the effect of betaNGF on spermatogenesis by using the systems of rat germ cell culture we established previously. betaNGF did not modify the number of Sertoli cells, pachytene spermatocytes, secondary spermatocytes nor the half-life of round spermatids, but increased the number of secondary meiotic metaphases and decreased the number of round spermatids formed in vitro. These effects of betaNGF were reversible and maximal at about 4 x 10(-11) M. Conversely, K252a, a Trk-specific kinase inhibitor, enhanced the number of round spermatids above that of control cultures. The presence of betaNGF and its receptors TrkA and p75(NTR) was investigated in testis sections, in Sertoli cell and germ cell fractions, and in germ cell and Sertoli cell co-cultures. betaNGF was detected only in germ cells from pachytene spermatocytes of stages VII up to spermatids of stages IX-X. TrkA and p75(NTR) were detected in Sertoli cells and in these germ cells. Taken together, these results indicate that betaNGF should participate in an auto/paracrine pathway of regulation of the second meiotic division of rat spermatocytes in vivo.     

Cytological and expression studies and quantitative analysis of the temporal and stage-specific effects of follicle-stimulating hormone and testosterone during cocultures of the normal human seminiferous epithelium

In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.     

Stimulation of rat Sertoli cell secretory activity in vitro by germ cells and residual bodies

The direct influence of germ cells and residual bodies on Sertoli cell basal and FSH-stimulated secretion of androgen-binding protein (ABP) was studied using Sertoli cells, recovered from 20-day-old rats, cultured alone or cocultured with a crude germ cell preparation from adult rats or with pachytene spermatocytes, round spermatids or populations of residual bodies enriched by centrifugal elutriation. The effect of a rat liver epithelial cell line (LEC) on Sertoli cell function was also tested. Addition of a crude germ cell preparation increased basal and FSH-stimulated ABP secretion. Pachytene spermatocytes and residual bodies adhered to the Sertoli cell monolayer to a much greater extent than did round spermatids. Addition of pachytene spermatocytes markedly enhanced basal and FSH-stimulated ABP secretion over 12 days of culture. Round spermatids and residual bodies stimulated ABP secretion although to a lesser extent than did spermatocytes. Furthermore, the increase of FSH-stimulated ABP levels was not maintained after 4 or 8 days of culture. LEC also enhanced basal and FSH-induced ABP levels but the increase of FSH-induced ABP production was only observed until Day 8 of culture. The influence of LEC on Sertoli cell secretion could be mediated through the production of an extracellular matrix. It is concluded that germ cells, particularly pachytene spermatocytes, can directly stimulate Sertoli cell secretory activity in vitro.     

Characterization of high mobility group protein levels during spermatogenesis in the rat

The distribution, quantitation, and synthesis of high mobility group (HMG) proteins during spermatogenesis in the rat have been determined. HMG1, -2, -14, and -17 were isolated from rat testes by Bio-Rex 70 chromatography combined with preparative gel electrophoresis. Amino acid analysis revealed that each rat testis HMG protein was similar to its calf thymus analogue. Tryptic peptide maps of somatic and testis HMG2 showed no differences and, therefore, failed to detect an HMG2 variant. Testis levels of HMG proteins, relative to DNA content, were equivalent to other tissues for HMG1 (13 micrograms/mg of DNA), HMG14 (3 micrograms/mg of DNA), and HMG17 (5 micrograms/mg of DNA). The testis was distinguished in that it contained a substantially higher level of HMG2 than any other rat tissue (32 micrograms/mg of DNA). HMG protein levels were determined from purified or enriched populations of testis cells representing the major stages of spermatogenesis; spermatogonia and early primary spermatocytes, pachytene spermatocytes, early spermatids, and late spermatids; and testicular somatic cells. High levels of HMG2 in the testis were due to pachytene spermatocytes and early spermatids (56 +/- 4 and 47 +/- 6 micrograms/mg of DNA, respectively). Mixtures of spermatogonia and early primary spermatocytes showed lower levels of HMG2 (12 +/- 3 micrograms/mg of DNA) similar to proliferating somatic tissues, whereas late spermatids had no detectable HMG proteins. The somatic cells of the testis, including isolated populations of Sertoli and Leydig cells, showed very low levels of HMG2 (2 micrograms/mg of DNA), similar to those in nonproliferating somatic tissues. HMG proteins were synthesized in spermatogonia and primary spermatocytes, but not in spermatids. Rat testis HMG2 exhibited two bands on acid-urea gels. A "slow" form comigrated with somatic cell HMG2, while the other "fast" band migrated ahead of the somatic form and appeared to be testis-specific. The "fast" form of HMG2 accounted for the large increase of HMG2 levels in rat testes. These results show that the very high level of HMG2 in testis is not associated with proliferative activity as previously hypothesized.     

Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures

Sertoli-spermatogenic cell co-cultures prepared from sexually immature rats (20-22 days old) and maintained in serum-free, hormone/growth factor-supplemented medium were used to determine the cell-specific localization of the growth factor somatomedin-C (SM-C). SM-C localization studies were carried out by indirect immunofluorescence using a monoclonal antibody (sm-1.2) to SM-C. In cultured rat hepatocytes, Sertoli and testicular peritubular cells, SM-C immunoreactivity was observed as a diffuse distribution of discrete immunofluorescent granules. Radio-immunoassay experiments using a rabbit antibody against human SM-C showed that testicular peritubular cells and Sertoli cells in primary culture accumulated SM-C in the medium. In spermatogenic cells co-cultured with subjacent Sertoli cells, immunoreactive SM-C was associated with pachytene spermatocytes but not with spermatogonia or early meiotic prophase spermatocytes (leptotene or zygotene). Both Sertoli cells and pachytene spermatocytes displayed binding sites for exogenously added SM-C. SM-C6 binding to spermatocytes reaching an advanced stage of meiotic prophase suggests a possible role of this growth factor in the meiotic process.