Novel class of glycosphingolipids involved in male fertility

Mice require testicular glycosphingolipids (GSLs) for proper spermatogenesis. Mutant mice strains deficient in specific genes encoding biosynthetic enzymes of the GSL pathway including Galgt1 (encoding GM2 synthase) and Siat9 (encoding GM3 synthase) have been established lacking various overlapping subsets of GSLs. Although male Galgt1-/- mice are infertile, male Siat9-/- mice are fertile. Interestingly, GSLs thought to be essential for male spermatogenesis are not synthesized in either of these mice strains. Hence, these GSLs cannot account for the different phenotypes. A novel class of GSLs was observed composed of eight fucosylated molecules present in fertile but not in infertile mutant mice. These GSLs contain polyunsaturated very long chain fatty acid residues in their ceramide moieties. GSLs of this class are expressed differentially in testicular germ cells. More importantly, the neutral subset of this new GSL class strictly correlates with male fertility. These data implicate polyunsaturated, fucosylated GSLs as essential for spermatogenesis and male mouse fertility.     

Mouse hybrid sterility and testicular function

Crosses of BALB/c female mice and inbred wild male mice (PWD, PWK) produce fertile female progeny, but the male offspring are sterile. The hybrid male sterility is a direct action of the hybrid sterility genes Hst-1s and Hstws. Previous reports concluded that spermatogenic arrest effected the sterility. However, the testicular steroidogenesis of hybrid sterile male mice has not been elucidated. In the present report, the steroidogenic capacity of hybrid sterile and parental strain males was directly assessed by quantifying testosterone secretion by maximally stimulated testes perfused in vitro. Additionally, Leydig cell mass and germ cell volumes were morphometrically determined. The experimental results confirm the deleterious impact of the Hst-1s/Hstws genotype on spermatogenesis and demonstrate for the first time that the steroidogenic capacity of hybrid sterile testes is reduced. The biochemical defects that cause the impairment of testicular function are unknown.     

Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate

Normal spermatogenesis is essential for reproduction and depends on proper spermatogonial stem cell (SSC) function. Genes and signaling pathways that regulate SSC function have not been well defined. We report that glial cell-line-derived neurotrophic factor (GDNF) signaling through the RET tyrosine kinase/GFRA1 receptor complex is required for spermatogonial self-renewal in mice. GFRA1 and RET expression was identified in a subset of gonocytes at birth, was restricted to SSCs during normal spermatogenesis, and RET expressing cells were abundant in a cryptorchid model of SSC self-renewal. We used the whole-testis transplantation technique to overcome the limitation of neonatal lethality of Gdnf-, Gfra1-, and Ret-deficient mice and found that each of these genes is required for postnatal spermatogenesis and not for embryological testes development. Each mutant testis shows severe SSC depletion by Postnatal Day 7 during the first wave of spermatogenesis. These defects were due to lack of SSC proliferation and an inability of SSCs to maintain an undifferentiated state. Our results demonstrate that GDNF-mediated RET signaling is critical for the fate of undifferentiated spermatogonia and that abnormalities in this pathway may contribute to male infertility and testicular germ cell tumors.     

Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis

Primordial germ cells (PGCs) are derived from a population of pluripotent epiblast cells in mice. However, little is known about when and how PGCs acquire the capacity to differentiate into functional germ cells, while keeping the potential to derive pluripotent embryonic germ cells and teratocarcinomas. In this investigation, we show that epiblast cells and PGCs can establish colonies of spermatogenesis after transfer into postnatal seminiferous tubules of surrogate infertile mice. Furthermore, we obtained normal fertile offspring by microinsemination using spermatozoa or spermatids derived from PGCs harvested from fetuses as early as 8.5 days post coitum. Thus, fetal male germ cell development is remarkably flexible, and the maturation process, from epiblast cells through PGCs to postnatal spermatogonia, can occur in the postnatal testicular environment. Primordial germ cell transplantation techniques will also provide a novel tool to assess the developmental potential of PGCs, such as those manipulated in vitro or recovered from embryos harboring lethal mutations.     

Effects of W (c-kit) gene mutation on gametogenesis in male mice: agametic tubular segments in Wf/Wf testes.

Mutations of the W (c-kit) gene, which encodes a transmembrane tyrosine kinase receptor, affect the development and differentiation of many types of stem cell. Most homozygous W mutant mice are sterile, due to a lack of germ cells arising during embryonic development, but one of the notable exceptions is Wf/Wf mice, which are fully fertile in both sexes. In order to elucidate the effects of the Wf mutation on spermatogenesis, postnatal spermatogenesis in Wf/Wf mice was histologically examined. The number of gonocytes at birth was significantly reduced and small portions of agametic seminiferous tubule segments were observed in mutant mice. It is suggested that this is due to a deficiency of primordial germ cells (PGC). Other than the agametic tubules, there was no evidence of reduced spermatogenesis after birth. These results indicate that the function of the W (c-kit) gene is more necessary for the development of PGC than for postnatal germ cells.     

RanBPM is essential for mouse spermatogenesis and oogenesis

RanBPM is a recently identified scaffold protein that links and modulates interactions between cell surface receptors and their intracellular signaling pathways. RanBPM has been shown to interact with a variety of functionally unrelated proteins; however, its function remains unclear. Here, we show that RanBPM is essential for normal gonad development as both male and female RanBPM(-/-) mice are sterile. In the mutant testis there was a marked decrease in spermatogonia proliferation during postnatal development. Strikingly, the first wave of spermatogenesis was totally compromised, as seminiferous tubules of homozygous mutant animals were devoid of post-meiotic germ cells. We determined that spermatogenesis was arrested around the late pachytene-diplotene stages of prophase I; surprisingly, without any obvious defect in chromosome synapsis. Interestingly, RanBPM deletion led to a remarkably quick disappearance of all germ cell types at around one month of age, suggesting that spermatogonia stem cells are also affected by the mutation. Moreover, in chimeric mice generated with RanBPM(-/-) embryonic stem cells all mutant germ cells disappeared by 3 weeks of age suggesting that RanBPM is acting in a cell-autonomous way in germ cells. RanBPM homozygous mutant females displayed a premature ovarian failure due to a depletion of the germ cell pool at the end of prophase I, as in males. Taken together, our results highlight a crucial role for RanBPM in mammalian gametogenesis in both genders.     

Size changes of protamine 1 mRNA provide a molecular marker to monitor spermatogenesis in wild-type and mutant mice

We utilized a cDNA encoding the cysteine-rich, tyrosine-containing mouse protamine, mouse protamine 1 (MP1), to detect the presence of several classes of differentiating germ cells in testicular extracts from wild-type and male sterile mutant mice. This assay is based on the changes in the poly (A) length of MP1-mRNA during spermatogenesis. Testicular extracts of sexually mature CD-1 mice contain a heterogeneous population of protamine-1 mRNA ranging in length from 450 to 580 nucleotides. When the protamine-1 probe was hybridized to testicular RNA preparations from 16- to 20-day-old animals, no MP1-mRNA was detected. Twenty-four-day-old mice contain only the 580-nucleotide form of MP1-mRNA. This size class of protamine mRNA is also present in purified populations of round spermatids, whereas elongating spermatids and residual bodies contain mRNAs ranging from 450 to 580 nucleotides in length, which are identical in size to those present in the testes of sexually mature animals. When the protamine cDNA probe was used to examine the progression of spermiogenesis in three male sterile mouse mutants, blind sterile (bs), quaking (qk) and testicular feminization (Tfm), the results demonstrated that each mutant is pathologically distinct. Analysis of the bs mutant revealed a diminution in the amount of both size classes of MP1-mRNA, in agreement with the cytological reports of reduced numbers of haploid spermatogenic cells in these animals. The presence of both size classes of protamine mRNA in the qk mutant indicates that germ-cell differentiation has proceeded at least to the step-12 spermatid in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of normal progeny by intracytoplasmic sperm injection following grafting of testicular tissue from cloned mice that died postnatally

Animal cloning by nuclear transfer has been successful in several species and was expected to become an alternative reproductive technique. Among the problems associated with this cloning technique, however, are its low success rate and high mortality of cloned animals even if they develop to term. Nuclear transfer has thus come to be considered too difficult to apply as a reproductive technique. The transplantation of male germ cells or pieces of testicular tissue has enabled the induction of spermatogenesis from fetal or postnatal male mice. In the present study, we examined whether functional male gametes could be obtained by the transplantation of pieces of testicular tissue from cloned mice that died immediately after birth with typical aberrant phenotypes, such as large offspring syndrome. Donor testicular tissues were retrieved from cloned mice that died postnatally and were transplanted into the testes of recipient nude mice. Two to three months after transplantation, the grafted donor testicular tissue had grown in the host testis, and histological analysis showed that spermatogenesis occurred within the graft. Intracytoplasmic sperm injection demonstrated that the testicular sperm generated in the grafted donor tissue were able to support full-term development of progeny. These results clearly showed that functional spermatogenesis could be induced by transplanting testicular tissue from cloned mice that died postnatally into recipient mice. The strategy presented here will be applicable to cloned animals of other species, because the xenografting of testicular tissue into mice has been demonstrated previously to be possible.     

Germ cell sex and cell cycle

Germ cells are the only cells in the body capable of transferring an individual's genetic and epigenetic information to the next generation. However, the developmental processes that provide the foundation for male and female germ line development and later gamete production are complex and poorly understood. In mice the primordial germ cells enter the bipotential gonad at E10.5 and, in response to the testicular or ovarian micro-environment, commit to spermatogenesis or oogenesis. This paper reviews progress in understanding the molecular processes underlying the early stages of male and female germ line development.     

Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.     

A testicular germ cell-associated serine-threonine kinase, MAK, is dispensable for sperm formation

A member of the mitogen-activated protein kinase superfamily, MAK, has been proposed to have an important role in spermatogenesis, since Mak gene expression is highly restricted to testicular germ cells. To assess the biological function of MAK, we have established MAK-deficient (Mak(-/-)) mice. Mak(-/-) mice developed normally, and no gross abnormalities were observed. Spermatogenesis of the Mak(-/-) mice was also intact, and most of the mice were fertile. However, Mak(-/-) male-derived litter sizes and their sperm motility in vitro were mildly reduced. These data show that function of MAK is not essential for spermatogenesis and male fertility.     

Lymphoid-specific helicase (HELLS) is essential for meiotic progression in mouse spermatocytes

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells(-/-) to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells(-/-), Hells(+/-), and Hells(+/+) mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells(-/-) than from Hells(+/+) mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells(-/-) mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells(-/-) spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.     

White-spotting mutations affect the regenerative differentiation of testicular germ cells: demonstration by experimental cryptorchidism and its surgical reversal.

The effect of white-spotting (W) mutations on differentiation of testicular germ cells was investigated by using experimental cryptorchidism and its surgical reversal. All mutant mice used in this study (Wv/+, Wsh/+, Wf/+ and Wf/Wf) showed normal fertility and well-ordered spermatogenesis, as in congenic +/+ mice. In the cryptorchid testis, which contains only type A spermatogonia as germ cells, the number and the proliferative activity of type A spermatogonia in mutant mice were comparable to +/+ mice. On the other hand, surgical reversal of the cryptorchid testis in mutants resulted in impaired regenerative differentiation of germ cells. Although complete recovery of spermatogenesis was observed in +/+ mice, testicular weight in Wsh/+, Wf/+ and Wf/Wf mice recovered to approximately 60-70% of intact levels, and some portions of seminiferous epithelium showed incomplete spermatogenesis. In Wv/+ mice, however, ability to recover the weight was completely lost, and only type A spermatogonia existed as germ cells in seminiferous tubules 3 mo after surgical reversal. These results suggest that W mutation affects the differentiation through type A spermatogonia to type B spermatogonia, indicating the functional significance of W (c-kit) in early spermatogenesis.     

Transplantation of male germ line stem cells restores fertility in infertile mice

Azoospermia or oligozoospermia due to disruption of spermatogenesis are common causes of human male infertility. We used the technique of spermatogonial transplantation in two infertile mouse strains, Steel (Sl) and dominant white spotting (W), to determine if stem cells from an infertile male were capable of generating spermatogenesis. Transplantation of germ cells from infertile Sl/Sld mutant male mice to infertile W/Wv or Wv/W54 mutant male mice restored fertility to the recipient mice. Thus, transplantation of spermatogonial stem cells from an infertile donor to a permissive testicular environment can restore fertility and result in progeny with the genetic makeup of the infertile donor male.     

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice

Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice overexpressing EF1alpha promoter-driven UCH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.     

Vitamin A deficiency results in meiotic failure and accumulation of undifferentiated spermatogonia in prepubertal mouse testis

Vitamin A (retinol) is required for maintenance of adult mammalian spermatogenesis. In adult rodents, vitamin A withdrawal is followed by a loss of differentiated germ cells within the seminiferous epithelium and disrupted spermatogenesis that can be restored by vitamin A replacement. However, whether vitamin A plays a role in the differentiation and meiotic initiation of germ cells during the first round of mouse spermatogenesis is unknown. In the present study, we found that vitamin A depletion markedly decreased testicular expression of the all-trans retinoic acid-responsive gene, Stra8, and caused meiotic failure in prepubertal male mice lacking lecithin:retinol acyltransferase (Lrat), encoding for the major enzyme in liver responsible for the formation of retinyl esters. Rather than undergoing normal differentiation, germ cells accumulated in the testes of Lrat(-/-) mice maintained on a vitamin A-deficient diet. These results, together with our previous observations that germ cells fail to enter meiosis and remain undifferentiated in embryonic vitamin A-deficient ovaries, support the hypothesis that vitamin A regulates the initiation of meiosis I of both oogenesis and spermatogenesis in mammals.     

The histone deacetylase SIRT1 controls male fertility in mice through regulation of hypothalamic-pituitary gonadotropin signaling

Sirtuins (SIRTs) are class-III NAD-dependent histone deacetylases (HDACs) that regulate various physiological processes. Inactivation of SIRT1 in the mouse leads to male sterility, but the molecular mechanisms responsible for this phenotype have not been determined. Here we show that fetal testis development appears normal in Sirt1(-/-) mice. In contrast, the first round of spermatogenesis arrests before the completion of meiosis with abundant apoptosis of pachytene spermatocytes, abnormal Leydig and Sertoli cell maturation, and strongly reduced intratesticular testosterone levels. We show that this phenotype is the consequence of diminished hypothalamic gonadotropin-releasing hormone expression and strongly reduced luteinizing hormone levels. Rather than having an intrinsic effect on male germ cells per se, our results show that SIRT1 regulates spermatogenesis at postnatal stages by controlling hypothalamus-pituitary gonadotropin (HPG) signaling. In addition to its well studied role in control of metabolism and energy homeostasis, our results thus reveal a novel and critical function of SIRT1 in controlling HPG signaling. This phenotype is more severe than those previously described using mice bred on different genetic backgrounds, and highlights the fact that SIRT1 function is strongly modified by other genetic loci.