The emerging role of mitochondrial derived peptide humanin in the testis

The discovery of mitochondrial derive peptides (MDPs) has spotlighted mitochondria as central hubs in control and regulation of cell viability and metabolism in the testis in response to intracellular and extracellular stresses. MDPs (Humanin, MOTS-c and SHLP-2) are present in testes. Humanin, the first MDP, is predominantly expressed in Leydig cells, and moderately in germ cells and seminal plasma. The administration of synthetic humanin peptide agonist HNG protects male germ cells against apoptosis induced by intratesticular hormonal deprivation, testicular hyperthermia, and chemotherapeutic agents in rodent testes. Humanin interacting with IGFBP-3 and/or Bax (pro-apoptotic proteins) prevents the activation of germ cell apoptosis. Humanin participates in the network of IL-12/IL-27 family of cytokines to exert the immune-modulation of the testicular environment. Humanin and other MDPs may be important in the amelioration of testicular stress and prevention of cell injury with possible implications for male infertility, fertility preservation and contraceptive development.     

Haploinsufficiency of Bcl-x leads to male-specific defects in fetal germ cells: differential regulation of germ cell apoptosis between the sexes

In germ cells, the function of which is to form the next generation, apoptotic cell death occurs during development, as in the case of somatic cells. In this study, we show that Bcl-x knockout heterozygous (Bcl-x(+/-)) mice exhibit severe defects in male germ cells during development. A substantial increase in apoptosis of male germ cells occurs at around embryonic day 13.5 (E13.5) in Bcl-x(+/-) embryos, leading to hypoplasia of postnatal testes and reduced fertility. On the other hand, female germ cells at the same stages do not show discernible differences between wild-type and Bcl-x(+/-) embryos. This phenotype of Bcl-x haploinsufficiency shows that regulation of apoptosis becomes different between the sexes at around the onset of sex differentiation. Through this study, we found that, in wild-type embryos, (1) apoptosis is much more frequent (approximately 10 times) in the male than in female germ cells, and (2) expression of Bcl-xL, but not that of Bax, is higher in female than in male germ cells, at around E13.5. Male fetal germ cells, cultured with gonadal somatic cells in vitro, showed higher frequencies of apoptosis than those cultured without gonadal somatic cells. On the other hand, in the absence of gonadal somatic cells, both male and female fetal germ cells in vitro showed similar frequencies of apoptosis to female fetal germ cells in vivo. Therefore, male germ cell apoptosis, of which the default pathway is similar to that of the female, is likely to be influenced by male gonadal environments.     

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.     

Regulation of human male germ cell death by modulators of ATP production

The understanding of testicular physiology, pathology, and male fertility issues requires knowledge of male germ cell death and energy production. Here, we induced human male germ cell apoptosis (detected by Southern blot analysis of DNA fragmentation, TUNEL, activation of caspases-3 and -9, and electron microscopy) by incubating seminiferous tubule segments under hormone- and serum-free conditions. Inhibitors of complexes I to IV of mitochondrial respiration, exposure to anoxia, and inhibition of F0F1-ATPase (with oligomycin) decreased the ATP levels (analyzed by HPLC) and suppressed apoptosis at 4 h. Uncoupler 2,4-dinitrophenol (DNP) and oligomycin combination also suppressed death at 4 h, as did the DNP alone. Inhibition of glycolysis by 2-deoxyglucose neither suppressed nor further induced apoptosis nor altered the antiapoptotic effects of the mitochondrial inhibitors. Furthermore, Fas system activation did not modify the effects of mitochondrial modulators. After 24 h, delayed male germ cell apoptosis was observed despite the presence of the mitochondrial inhibitors. We conclude that the mitochondrial ATP production machinery plays an important role in regulating in vitro-induced primary pathways of human male germ apoptosis. The ATP synthesized by the F0F1-ATPase seems to be the crucial death regulator, rather than any of the complexes (I-IV) alone, the functional electron transport chain, or the membrane potential. We also conclude that there seem to be secondary pathways of human testicular cell apoptosis that do not require mitochondrial ATP production. The present study emphasizes the role of the main catabolic pathways in the complex network of regulating events of male germ cell life and death.     

Clinical aspects of male germ cell apoptosis during testis development and spermatogenesis

Apoptosis appears to have an essential role in the control of germ cell number in testes. During spermatogenesis germ cell deletion has been estimated to result in the loss of up to 75% of the potential number of mature sperm cells. At least three factors seem to determine the onset of apoptosis in male germ cells: (1) lack of hormones, especially gonadotropins and androgens; (2) the specific stage in the spermatogenic cycle; (3) and the developmental stage of the animal. Although male germ cell apoptosis has been well characterized in various animal models, few studies are presently available regarding germ cell apoptosis in the human testis. The first part of this review is focused on germ cell apoptosis in testes of prepubertal boys, with special emphasis on apoptosis in normal and cryptorchid testes. A higher percentage of apoptotic spermatogonia was seen in the cryptorchid testes than in the scrotal testes. The hCG-treatment increased the number of apoptotic spermatogonia. The hCG-treatment-induced apoptosis in spermatogonia had severe long-term consequences in reproductive functions in adulthood. Increased apoptosis after hCG-treatment was associated with subnormal testis volumes, subnormal sperm density and pathologically elevated serum FSH. This finding indicates that increased apoptosis in spermatogonia in prepuberty leads to disruption of testis development. To evaluate the role of apoptosis in human adult testes, apoptosis was induced in seminiferous tubules that were incubated under serum-free conditions in the absence or presence of testosterone. Most frequently apoptosis was identified in spermatocytes. Occasionally some spermatids also showed signs of apoptosis. In short term incubations apoptosis was suppressed by testosterone. Our findings lead to the conclusion that apoptosis is a normal, hormonally controlled phenomenon in the human testis. The role of apoptosis in disorders of spermatogenesis remains to be established.     

Sdmg1 is a conserved transmembrane protein associated with germ cell sex determination and germline-soma interactions in mice

In mammals, the supporting cell lineage in an embryonic gonad communicates the sex-determining decision to various sexually dimorphic cell types in the developing embryo, including the germ cells. However, the molecular nature of the sex-determining signals that pass from the supporting cells to the germ cells is not well understood. We have identified a conserved transmembrane protein, Sdmg1, owing to its male-specific expression in mouse embryonic gonads. Sdmg1 is expressed in the Sertoli cells of embryonic testes from 12.5 dpc, and in granulosa cells of growing follicles in adult ovaries. In Sertoli cells, Sdmg1 is localised to endosomes, and knock-down of Sdmg1 in Sertoli cell lines causes mis-localisation of the secretory SNARE Stx2 and defects in membrane trafficking. Upregulation of Sdmg1 appears to be part of a larger programme of changes to membrane trafficking pathways in embryonic Sertoli cells, and perturbing secretion in male embryonic gonads in organ culture causes male-to-female germ cell sex reversal. These data suggest that changes that occur in the cell biology of embryonic Sertoli cells may facilitate the communication of male sex-determining decisions to the germ cells during embryonic development.     

Male infertility due to germ cell apoptosis in mice lacking the thiamin carrier, Tht1. A new insight into the critical role of thiamin in spermatogenesis

A mouse model of thiamin-responsive megaloblastic anemia (diabetes mellitus, deafness, megaloblastic anemia) lacking functional Slc19a2 has been generated and unexpectedly found to have a male-specific sterility phenotype. We describe here the characterization of the testis-specific effects of absence of the high-affinity thiamin transporter, Tht1. Null males were found to have hypoplastic testes secondary to germ cell depletion. Morphologic and expression analysis revealed that under conditions of standard thiamin intake, tissues affected in the syndrome (pancreatic beta-cell, hematopoietic cells, auditory nerve) maintained normal function but pachytene stage spermatocytes underwent apoptosis. Under conditions of thiamin challenge, the apoptotic cell loss extended to earlier stages of germ cells but spared Sertoli cells and Leydig cells. Injection of high-dose thiamin was effective in reversing the spermatogenic failure, suggesting that the absence of the thiamin carrier could be overcome by diffusion-mediated transport at supranormal thiamin concentrations. These observations demonstrated that male germ cells, particularly those with high thiamin transporter expression beyond the blood-testis barrier, were more susceptible to apoptosis triggered by intracellular thiamin deficiency than any other tissue type. The findings described here highlight an unexpected and critical role for thiamin transport and metabolism in spermatogenesis.     

Age-dependent loss of sperm production in mice via impaired lysophosphatidic acid signaling

Approximately half of all infertility cases can be attributed to male reproductive dysfunction for which low sperm count is a major contributing factor. The current study identified receptor-mediated lysophosphatidic acid (LPA) signaling as a new molecular component influencing male fertility. LPA is a small signaling phospholipid, the effects of which are mediated through at least five G protein-coupled receptors, named LPA 1-5. LPA1/2/3, but not LPA4/5, show high expression in mouse testis. Mice deficient in LPA1/2/3 showed a testosterone-independent reduction of mating activity and sperm production, with an increased prevalence of azoospermia in aging animals. A significant increase of germ cell apoptosis also was observed in testes. Germ cell apoptosis led to a reduction in germ cell proliferation. These data demonstrate a novel in vivo function for LPA signaling as a germ cell survival factor during spermatogenesis.     

Sertoli cell development and function in an animal model of testicular dysgenesis syndrome

Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by approximately 50% at E21.5 but recovered to normal by Days 25-90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-müllerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells.     

CD9 is expressed on human male germ cells that have a long-term repopulation potential after transplantation into mouse testes

Human spermatogonial stem cells (SSCs) play critical roles in lifelong maintenance of male fertility and regeneration of spermatogenesis. These cells are expected to provide an important resource for male fertility preservation and restoration. A basic strategy has been proposed that would involve harvesting testis biopsy specimens from a cancer patient prior to cancer therapies, and transplanting them back to the patient at a later time; then, SSCs included in the specimens would regenerate spermatogenesis. To clinically apply this strategy, isolating live human SSCs is important. In this study, we investigated whether CD9, a known rodent SSC marker, is expressed on human male germ cells that can repopulate recipient mouse testes upon transplantation. Testicular tissues were obtained from men with obstructive azoospermia. Using immunohistochemistry, we found that CD9 was expressed in human male germ cells in the basal compartment of the seminiferous epithelium. Following immunomagnetic cell sorting, CD9-positive cells were enriched for germ cells expressing MAGEA4, which is expressed by spermatogonia and some early spermatocytes, compared with unsorted cells. We then transplanted CD9-positive cells into nude mouse testes and detected an approximately 3- to 4-fold enrichment of human germ cells that repopulated mouse testes for at least 4 mo after transplantation, compared with unsorted cells. We also observed that some cell turnover occurred in human germ cell colonies in recipient testes. These results demonstrate that CD9 identifies human male germ cells with capability of long-term survival and cell turnover in the xenogeneic testis environment.     

Prognostic value of germ cells in the ejaculate: a case study

Abstract

The first approach to assessing male fertility is to study a spermiogram, where special attention is given to sperm count, motility and morphology, while less attention is given to other cells in the ejaculate. Normal spermatogenesis requires a balance between cell death and proliferation; therefore, the number of germ cells (GC) in the ejaculate is less than the number of sperm. We propose a new index for altered spermatogenesis, i.e., the rate GC/sperm. We investigated a patient with oligozoospermia and a GC/sperm ratio greater than one, which indicated that spermatogenesis had been damaged. Complementary cytological tests were employed to characterized GC status: Papanicolaou stain, transmission electronic microscopy (TEM), vitality test, AgNOR and TUNEL assay. We also correlated cell morphology with ultrastructure studies that showed apoptosis. Nuclear apoptosis is characterized by vacuolization, misshapen nuclei, and “half moon,” dispersed, uncondensed, disrupted and smudged chromatin. Cytoplasmic apoptosis is characterized by vacuolization, cytoplasmic protrusions, lamellar bodies, and swollen endoplasmic reticulum and mitochondria. To date, only testicular biopsy has been used to diagnose complete or incomplete testicular arrest. Our investigation is the first to determine a cytological feature in semen samples that could be used as a biological marker for abnormal spermatogenesis and for predicting the transition from oligospermia to azoospermia.     

Protection from radiation-induced male germ cell loss by sphingosine-1-phosphate

Male germ cells are susceptible to radiation-induced injury, and infertility is a common problem after total-body irradiation. Here we investigated, first, the effects of irradiation on germ cells in mouse testis and, second, the role of sphingosine-1-phosphate (S1P) treatment in radiation-induced male germ cell loss. Irradiation of mouse testes mainly damaged the early developmental stages of spermatogonia. The damage was seen by means of DNA flow cytometry 21 days after irradiation as decreasing numbers of spermatocytes and spermatids with increasing amounts of ionizing radiation (0.1-2.0 Gy). Intratesticular injections of S1P given 1-2 h before irradiation (0.5 Gy) did not protect against short-term germ cell loss as measured by in situ end labeling of DNA fragmentation 16 h after irradiation. However, after 21 days, in the S1P-treated testes, the numbers of primary spermatocytes and spermatogonia at G2 (4C peak as measured by flow cytometry) were higher at all stages of spermatogenesis compared with vehicle-treated testes, indicating protection of early spermatogonia by S1P, whereas the spermatid (1C) populations were similar. In conclusion, S1P appears to protect partially (16%-47%) testicular germ cells against radiation-induced cell death. This warrants further studies aimed at development of therapeutic agents capable of blocking sphingomyelin-induced pathways of germ cell loss.