HST-1/FGF-4 protects male germ cells from apoptosis under heat-stress condition

Apoptosis plays an important role in controlling the number of male germ cells and eliminating defective germ cells during testicular development and spermatogenesis. We show here that fibroblast growth factor-4 (HST-1/FGF-4) may play a critical role as a survival factor for germ cells, protecting them from apoptosis. Testes of adult male mice that received an adenovirus carrying human HST-1/FGF-4 (AxHST-1) or a control adenovirus (AxCAwt) were exposed to mild hyperthermia, which causes germ cell apoptosis. An in situ terminal-deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling (TUNEL) assay characterized germ cell apoptosis. The results indicated that HST-1/FGF-4 significantly reduced the apoptotic death of germ cells and prevented testicular weight loss and sperm count reduction. We also found that Hst-1/Fgf-4 present in testes is up-regulated in vivo when the testes are exposed to mild hyperthermia, and that endogenous Hst-1/Fgf-4 mRNA expression in Sertoli cells are also induced when the cells are exposed to mild hyperthermia in vitro. In addition, the MAPK cascade, which could increase an FGF-dependent survival signal, is activated by HST-1/FGF-4 stimuli in germ cells. On the other hand, upon HST-1/FGF-4 stimulation, lactate production from Sertoli cells were induced, which is indispensable nutrient for germ cell survival. These results suggest that HST-1/FGF-4 can act as an important physiological anti-apoptotic factor for male germ cells in stimulating lactate production of Sertoli cells upon heat stress, thereby promoting germ cell survival.     

Effect of metformin on testicular expression and localization of leptin receptor and levels of leptin in the diabetic mice

Diabetes mellitus impairs testicular activity and leads to infertility. Leptin is one of the endogenous regulators of the male reproductive functions, but the role of leptin and its receptor (LEPR/Ob‐R) in the control of testosterone production and testicular proliferation has not been investigated so far, especially in the Type 1 diabetes mellitus (DM1). Metformin is an anti‐hyperglycemic drug which is beneficial for treating the both DM2 and DM1. The aim of this work was to study the possible role of leptin and Ob‐R in the regulation of steroidogenesis and proliferation in the testes of mice with streptozotocin‐induced DM1 (75 mg/kg/day, 4 days) and to estimate the restoring effect of metformin treatment (500 mg/kg, 2 weeks) on the diabetic testes. In the diabetic testes, the plasma and intratesticular leptin levels and plasma testosterone levels were reduced and completely restored by metformin treatment. Metformin also restored the expression of the steroidogenic transport protein steroidogenic acute regulatory protein reduced in DM1. In the diabetic testes, the expression of Ob‐R was downregulated and the immunolocalization of Ob‐R showed weak staining in the Leydig cells, the primary spermatocytes and the round spermatids. The germ cell proliferation was also reduced in DM1, as noticed with proliferating cell nuclear antigen (PCNA) expression. Metformin increased the Ob‐R expression and immunostaining in the different cell types and improved the PCNA expression. Thus, DM1 impairs the testicular steroidogenesis and proliferation by inhibiting the leptin signaling, causing a decrease in leptin levels and Ob‐R expression in the testes of diabetic mice, while metformin improves the leptin signaling and restores testosterone production and testicular proliferation.     

Chromosome X modulates incidence of testicular germ cell tumors in <Emphasis Type="Italic">Ter</Emphasis> mice

Germ cell tumor development in humans has been proposed to be part of testicular dysgenesis syndrome (TDS), which manifests as undescended testes, sterility, hypospadias, and, in extreme cases, as germ cell tumors. Males of the Ter mouse strain show interesting parallels to TDS because they either lack germ cells and are sterile or develop testicular germ cell tumors. We found that these defects in Ter mice are due to mutational inactivation of the Dead-end (Dnd1) gene. Here we report that chromosome X modulates germ cell tumor development in Ter mice. We tested whether the X or the Y chromosome influences tumor incidence. We used chromosome substitution strains to generate two new mouse strains: 129-Ter/Ter that carry either a C57BL/6J (B6)-derived chromosome (Chr) X or Y. We found that Ter/Ter males with B6-Chr X, but not B6-Chr Y, showed a significant shift in propensity from testicular tumor development to sterile testes phenotype. Thus, our studies provide unambiguous evidence that genetic factors from Chr X modulate the incidence of germ cell tumors in mice with inactivated Dnd1. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

A novel system of fertility rescue in Drosophila hybrids reveals a link between hybrid lethality and female sterility

Hybrid daughters of crosses between Drosophila melanogaster females and males from the D. simulans species clade are fully viable at low temperature but have agametic ovaries and are thus sterile. We report here that mutations in the D. melanogaster gene Hybrid male rescue (Hmr), along with unidentified polymorphic factors, rescue this agametic phenotype in both D. melanogaster/D. simulans and D. melanogaster/D. mauritiana F(1) female hybrids. These hybrids produced small numbers of progeny in backcrosses, their low fecundity being caused by incomplete rescue of oogenesis as well as by zygotic lethality. F(1) hybrid males from these crosses remained fully sterile. Hmr(+) is the first Drosophila gene shown to cause hybrid female sterility. These results also suggest that, while there is some common genetic basis to hybrid lethality and female sterility in D. melanogaster, hybrid females are more sensitive to fertility defects than to lethality.     

Male sterility caused by p6H and qk mutations is not corrected in chimeric mice

It is not known if the male sterility caused by the pleiotropic mutations p6H (pink-eyed 6H) and qk (quaking) is intrinsic or extrinsic to spermatogenic cells. This question was addressed by juxtaposing mutant and normal cells in the testes of chimeric mice and determining whether the mutant germ cells could form functional sperm. Twenty-one male chimeras consisting of normal cells and p6H/p6H or qk/qk cells were analyzed. For each, breeding productivity and testicular and sperm morphology were determined. Karyotypes and isozyme analyses were performed to identify the two cellular components of each chimera. All male chimeras that contained p6H/p6H, XY cells were sterile. Although some chimeras with a qk/qk, XY mutant component were fertile, none produced offspring from the homozygous qk component. Spermatids of the sterile chimeras showed abnormalities characteristic of the mutations. We conclude from this study that the presence of normal XY germ and somatic cells in the testis did not rescue the male sterile phenotype of homozygous p6H or qk XY germ cells. Therefore, the action of these mutant genes in causing sperm abnormalities and sterility is autonomous to the germ cells.     

Polymorphism in hybrid male sterility in wild-derived Mus musculus musculus strains on proximal chromosome 17

The hybrid sterility-1 (Hst1) locus at Chr 17 causes male sterility in crosses between the house mouse subspecies Mus musculus domesticus (Mmd) and M. m. musculus (Mmm). This locus has been defined by its polymorphic variants in two laboratory strains (Mmd genome) when mated to PWD/Ph mice (Mmm genome): C57BL/10 (carrying the sterile allele) and C3H (fertile allele). The occurrence of sterile and/or fertile (wild Mmm × C57BL)F₁ males is evidence that polymorphism for this trait also exists in natural populations of Mmm; however, the nature of this polymorphism remains unclear. Therefore, we derived two wild-origin Mmm strains, STUS and STUF, that produce sterile and fertile males, respectively, in crosses with C57BL mice. To determine the genetic basis underlying male fertility, the (STUS × STUF)F₁ females were mated to C57BL/10 J males. About one-third of resulting hybrid males (33.8%) had a significantly smaller epididymis and testes than parental animals and lacked spermatozoa due to meiotic arrest. A further one-fifth of males (20.3%) also had anomalous reproductive traits but produced some spermatozoa. The remaining fertile males (45.9%) displayed no deviation from values found in parental individuals. QTL analysis of the progeny revealed strong associations of male fitness components with the proximal end of Chr 17, and a significant effect of the central section of Chr X on testes mass. The data suggest that genetic incompatibilities associated with male sterility have evolved independently at the proximal end of Chr 17 and are polymorphic within both Mmd and Mmm genomes.     

Effects on spermiogenesis in the mouse of a male sterile neurological mutation,Purkinje cell degeneration

Purkinje cell degeneration (pcd) is a neurological mutation in the mouse that causes male sterility, but not female sterility. In order to assess the effects of this mutation on spermiogenesis, the structure of the testis and of epididymal spermatozoa was examined by transmission and scanning electron microscopy. In the mutant males, the sperm count was reduced, sperm were nonmotile, and 93% of the sperm were characterized by structural abnormalities of the head, the tail, or both. In the testes of mutant mice, Sertoli cell structure was normal, as were also the early stages of spermiogenesis. However, the elongating and maturing spermatids were characterized by abnormally shaped heads and tails with extraneous and ectopic outer dense fibers. These defects were common in the testes of the mutant mice and rare in the testes of the littermate control mice. It was concluded that the structural abnormalities of the pcd sperm occurred during spermiogenesis and were not due to degeneration of the sperm in the epididymis. These structural abnormalities are similar to those found in all other reported male sterile mutants of the mouse; therefore, although they are caused by the expression of the pcd gene, they are not unique to the expression of this gene.     

Genetics of Hybrid Inviability and Sterility in Drosophila: Dissection of Introgression of D. simulans Genes in D. melanogaster Genome

Interspecific crosses between Drosophila melanogaster and Drosophila simulans usually produce sterile unisexual hybrids. The barrier preventing genetic analysis of hybrid inviability and sterility has been taken away by the discovery of a D. simulans strain which produces fertile female hybrids. D. simulans genes in the cytological locations of 21A1 to 22C1-23B1 and 30F3-31C5 to 36A2-7 have been introgressed into the D. melanogaster genetic background by consecutive backcrosses. Flies heterozygous for the introgression are fertile, while homozygotes are sterile both in females and males. The genes responsible for the sterility have been mapped in the introgression. The male sterility is caused by the synergistic effect of multiple genes, while the female sterility genes have been localized to a 170 kb region (32D2 to 32E4) containing 20 open reading frames. Thus, the female sterility might be attributed to a single gene with a large effect. We have also found that the Lethal hybrid rescue mutation which prevents the inviability of male hybrids from the cross of D. melanogaster females and D. simulans males cannot rescue those carrying the introgression, suggesting that D. simulans genes maybe non-functional in this hybrid genotype. The genes responsible for the inviability have not been separated from the female sterility genes by recombination.     

Hst-3: an X-linked hybrid sterility gene

A gene, Hst-3, responsible for sterility in F1 males from crosses between Mus spretus and laboratory strains of mice such as C57BL/6, has been localized on the distal part of the X chromosome, using both DNA probes and biochemical markers on a panel of F1(C57BL/6 x SEG) x C57BL/6 backcross males. This gene may be a model for studying mammalian hybrid sterility.     

Oocytes in newborn MRL mouse testes

Although mammals produce either sperm or eggs depending on their sex, we found oocytes in the testes of newborn MRL/MpJ male mice. In the present study, we report the morphological characteristics of testicular oocytes, the postnatal change of oocyte number per testis, and the expression of a few oocyte-specific genes in the testes of MRL/MpJ mice. The testicular oocytes had a diameter of 50-70 microm and were surrounded by zonae pellucidae, which were observed between oocytes and follicular epithelial cells. Ultrastructurally, the testicular oocytes contained numerous microvilli and cortical granules, receiving cytoplasmic projections from follicular epithelial cells. The testicular oocytes appeared as early as at birth, and the largest number was found on Day 14. The testicular oocytes were detected in only MRL strains and B6MRLF1, but not in C57BL/6, C3H/He, BALB/c, DBA/2, A/J, and MRLB6F1. The expression of the oocyte-specific genes Zp1, Zp2, Zp3, and Omt2a was detected in testes from MRL/MpJ mice. These results suggest that newborn male MRL/MpJ mice with XY chromosomes can produce oocytes in their testes and that one of the genes causing this exists on the Y chromosome.     

Sex-linked hybrid sterility in a butterfly

Recent studies, primarily in Drosophila, have greatly advanced our understanding of Haldane's rule, the tendency for hybrid sterility or inviability to affect primarily the heterogametic sex (Haldane 1922). Although dominance theory (Turelli and Orr 1995) has been proposed as a general explanation of Haldane's rule, this remains to be tested in female-heterogametic taxa, such as the Lepidoptera. Here we describe a novel example of Haldane's rule in Heliconius melpomene (Lepidoptera; Nymphalidae). Female F1 offspring are sterile when a male from French Guiana is crossed to a female from Panama, but fertile in the reciprocal cross. Male F1s are fertile in both directions. Similar female F1 sterility occurs in crosses between French Guiana and eastern Colombian populations. Backcrosses and linkage analysis show that sterility results from an interaction between gene(s) on the Z chromosome of the Guiana race with autosomal factors in the Panama genome. Large X (or Z) effects are commonly observed in Drosophila, but to our knowledge have not been previously demonstrated for hybrid sterility in Lepidoptera. Differences in the abundance of male versus female or Z-linked versus autosomal sterility factors cannot be ruled out in our crosses as causes of Haldane's rule. Nonetheless, the demonstration that recessive Z-linked loci cause hybrid sterility in a female heterogametic species supports the contention that dominance theory provides a general explanation of Haldane's rule (Turelli and Orr 2000).     

Hybrid Dysgenesis in DROSOPHILA MELANOGASTER: The Biology of Female and Male Sterility

High levels of female and male sterility were observed among the hybrids from one of the two reciprocal crosses between a wild strain of D. melanogaster known as pi2 and laboratory strains. The sterility, which is part of a common syndrome called hybrid dysgenesis, was found to be associated with the rudimentary condition of one or both of the ovaries or testes. All other tissues, including those of the reproductive system were normal, as were longevity and mating behavior. The morphological details of the sterility closely mimic the agametic condition occurring when germ cells are destroyed by irradiation or by the maternal-effect mutation, grandchildless. We suggest that sterility in hybrid dysgenesis is also caused by failure in the early development of germ cells. There is a thermo-sensitive period beginning at approximately the time of initiation of mitosis among primordial germ cells a few hours before the egg hatches and ending during the early larval stages. Our results suggest that hybrid dysgenesis, which also includes male recombination, mutation and other traits, may be limited to the germ line, and that each of the primordial germ cells develops, or fails to develop, independently of the others. This hypothesis is consistent with the observed frequencies of unilateral and bilateral sterility, with the shape of the thermosensitivity curves and with the fact that males are less often sterile than females. The features of this intraspecific hybrid sterility are found to resemble those seen in some interspecific Drosophila hybrids, especially those from the cross D. melanogaster X D. simulans.     

The Genetics of Reproductive Isolation in the Drosophila Simulans Clade: X Vs. Autosomal Effects and Male Vs. Female Effects

A strong effect of homozygous autosomal regions on reproductive isolation was found for crosses between the species in the Drosophila simulans clade. Second chromosome regions were introgressed from D. mauritiana and D. sechellia into D. simulans and tested for their homozygous effects on hybrid male and hybrid female sterility and inviability. Most introgressions are fertile as heterozygotes, yet produce sterile male offspring when made homozygous. The density of homozygous autosomal factors contributing to hybrid male sterility is comparable to the density of X chromosome factors for this level of resolution. Female sterility was also revealed, yet the disparity between male and female levels of sterility was great, with male sterility being up to 23 times greater than female sterility. Complete hybrid inviability was also associated with some regions of the second chromosome, yet there were no strong sex differences. In conclusion, we find no evidence to support a strong X chromosome bias in the evolution of hybrid sterility or inviability but do find a very strong sex bias in the evolution of hybrid sterility. In light of these findings, we reevaluate the current models proposed to explain the genetic pattern of reproductive isolation.     

Genetic complexity underlying hybrid male sterility in Drosophila

Recent genetic analyses of closely related species of Drosophila have indicated that hybrid male sterility is the consequence of highly complex synergistic effects among multiple genes, both conspecific and heterospecific. On the contrary, much evidence suggests the presence of major genes causing hybrid female sterility and inviability in the less-related species, D. melanogaster and D. simulans. Does this contrast reflect the genetic distance between species? Or, generally, is the genetic basis of hybrid male sterility more complex than that of hybrid female sterility and inviability? To clarify this point, the D. simulans introgression of the cytological region 34D-36A to the D. melanogaster genome, which causes recessive male sterility, was dissected by recombination, deficiency, and complementation mapping. The 450-kb region between two genes, Suppressor of Hairless and snail, exhibited a strong effect on the sterility. Males are (semi-)sterile if this region of the introgression is made homozygous or hemizygous. But no genes in the region singly cause the sterility; this region has at least two genes, which in combination result in male sterility. Further, the males are less fertile when heterozygous with a larger introgression, which suggests that dominant modifiers enhance the effects of recessive genes of male sterility. Such an epistatic view, even in the less-related species, suggests that the genetic complexity is special to hybrid male sterility.     

In-vitro studies of the development of pituitary and testicular functions in diabetes (C57Bl/KsJ-db/db) mutant mice

The hypothesis that male diabetes mutant mice (C57Bl/KsJ-db/db) are suffering from impairment of testicular steroidogenic function and pituitary LH release was tested. A smaller postpubertal increase of testicular weight and a reduction of plasma testosterone and androstenedione levels by 65% at 17 weeks of age were most obvious from the comparison to homozygous lean controls. The ability of constant amounts of Leydig cells, either in crude interstitial cell or in purified Leydig cell suspensions, to respond to maximal doses of hCG or cyclic AMP-was reduced by at least 40% in adult diabetes mice. This defect could be attributed to a 40% decrease of steroid-17 alpha-monooxygenase activity as compared to lean mice. No differences occurred, however, if Leydig cells were submaximally stimulated. GnRH-stimulated pituitary LH release was not significantly changed. The impairment of testicular steroidogenic function in diabetes mutant mice may represent a further aspect of infertility of these animals and of diabetes mellitus.     

Genetic studies of two sister species in the Drosophila melanogaster subgroup, D. yakuba and D. santomea

We performed genetic analysis of hybrid sterility and of one morphological difference (sex-comb tooth number) on D. yakuba and D. santomea, the former species widespread in Africa and the latter endemic to the oceanic island of S?o Tomé, on which there is a hybrid zone. The sterility of hybrid males is due to at least three genes on the X chromosome and at least one on the Y, with the cytoplasm and large sections of the autosomes having no effect. F1 hybrid females carrying two X chromosomes from either species are perfectly fertile despite their genetic similarity to completely sterile F1 hybrid males. This implies that the appearance of Haldane's rule in this cross is at least partially due to the faster accumulation of genes causing male than female sterility. The larger effects of the X and Y chromosomes than of the autosomes, however, also suggest that the genes causing male sterility are recessive in hybrids. Some female sterility is also seen in interspecific crosses, but this does not occur between all strains. This is seen in pure-species females inseminated by heterospecific males (probably reflecting incompatibility between the sperm of one species and the female reproductive tract of the other) as well as in inseminated F1 and backcross females, probably reflecting genetically based incompatibilities in hybrids that affect the reproductive system. The latter 'innate' sterility appears to involve deleterious interactions between D. santomea chromosomes and D. yakuba cytoplasm. The difference in male sex-comb tooth number appears to involve fairly large effects of the X chromosome. We discuss the striking evolutionary parallels in the genetic basis of sterility, in the nature of sexual isolation, and in morphological differences between the D. santomea/D. yakuba divergence and two other speciation events in the D. melanogaster subgroup involving island colonization.     

Influence of neonatal androgenization on the testicular steroidogenesis in the adult rat

The in vitro testicular steroidogenesis of male rats, androgenized on the third postnatal day by a single injection of 1 mg testosterone propionate, was investigated when the animals were 100 days old. The neonatal androgenization resulted in a 25% lower testes weight, significantly increased plasma levels of FSH (P less than 0.01) and LH (P less than 0.02), and normal levels of testosterone. Although the testes were hypotrophic, the incubation of the testes pairs yielded the same amounts of testosterone, 7 alpha-hydroxytestosterone and 5 alpha-androstane-(3 alpha + 3 beta), 17 beta-diol as in the control animals. However, the steroidogenic response to an acute hCG stimulation was reduced. From incubations of testes homogenates with various labelled steroid precursors it could be inferred that the activity of the 17 alpha-hydroxylase, the 3 beta-hydroxysteroid dehydrogenase-isomerase and the 17 beta-hydroxysteroid dehydrogenase, expressed per unit of incubated protein, was significantly increased in the testes of the androgenized rats. These data indicate that the basal steroidogenesis in neonatally androgenized male rats is maintained by an increased synthesis per unit of tissue, possibly under influence of an increased gonadotrophic stimulus, but that the maximum steroidogenic capacity is reduced.