Evolutionarily conserved mammalian adenine nucleotide translocase 4 is essential for spermatogenesis

The adenine nucleotide translocases (Ant) facilitate the transport of ADP and ATP by an antiport mechanism across the inner mitochondrial membrane, thus playing an essential role in cellular energy metabolism. We recently identified a novel member of the Ant family in mouse, Ant4, of which gene configuration as well as amino acid homology is well conserved among mammals. The conservation of Ant4 in mammals, along with the absence of Ant4 in nonmammalian species, suggests a unique and indispensable role for this ADP/ATP carrier in mammalian development. Of interest, in contrast to its paralog Ant2, which is encoded by the X chromosome and ubiquitously expressed in somatic cells, Ant4 is encoded by an autosome and selectively expressed in testicular germ cells. Immunohistochemical examination as well as RNA expression analysis using separated spermatogenic cell types revealed that Ant4 expression was particularly high in spermatocytes. When we generated Ant4-deficient mice by targeted disruption, a significant reduction in testicular size was observed without any other distinguishable abnormalities in the mice. Histological examination as well as stage-specific gene expression analysis in adult and neonatal testes revealed a severe reduction of spermatocytes accompanied by increased apoptosis. Subsequently, the Ant4-deficient male mice were infertile. Taken together, these data elucidated the indispensable role of Ant4 in murine spermatogenesis. Considering the unique conservation and chromosomal location of the Ant family genes in mammals, the Ant4 gene may have arisen in mammalian ancestors and been conserved in mammals to serve as the sole and essential mitochondrial ADP/ATP carrier during spermatogenesis where the sex chromosome-linked Ant2 gene is inactivated.     

The effects of progesterone on sexual behavior in male green anole lizards (Anolis carolinensis).

It is well known that androgen-dependent sexual behaviors in male mammals and birds are inhibited by exogenous progesterone (P). However, recent research on male whiptail lizards (Cnemidophorus inornatus) indicates that P can stimulate sexual and copulatory behavior. We report here both antiandrogenic and synandrogenic actions of P on sexual behavior in males of another reptile, the green anole lizard (Anolis carolinensis). Earlier reports on birds and mammals are reviewed and discussed in relation to a possible physiological role of P in influencing sexual behavior in male vertebrates.     

Identification and Comparative Analysis of the Protocadherin Cluster in a Reptile,the Green Anole Lizard

Background

The vertebrate protocadherins are a subfamily of cell adhesion molecules that are predominantly expressed in the nervous system and are believed to play an important role in establishing the complex neural network during animal development. Genes encoding these molecules are organized into a cluster in the genome. Comparative analysis of the protocadherin subcluster organization and gene arrangements in different vertebrates has provided interesting insights into the history of vertebrate genome evolution. Among tetrapods, protocadherin clusters have been fully characterized only in mammals. In this study, we report the identification and comparative analysis of the protocadherin cluster in a reptile, the green anole lizard (Anolis carolinensis).

Methodology/Principal Findings

We show that the anole protocadherin cluster spans over a megabase and encodes a total of 71 genes. The number of genes in the anole protocadherin cluster is significantly higher than that in the coelacanth (49 genes) and mammalian (54–59 genes) clusters. The anole protocadherin genes are organized into four subclusters: the δ, α, β and γ. This subcluster organization is identical to that of the coelacanth protocadherin cluster, but differs from the mammalian clusters which lack the δ subcluster. The gene number expansion in the anole protocadherin cluster is largely due to the extensive gene duplication in the γb subgroup. Similar to coelacanth and elephant shark protocadherin genes, the anole protocadherin genes have experienced a low frequency of gene conversion.

Conclusions/Significance

Our results suggest that similar to the protocadherin clusters in other vertebrates, the evolution of anole protocadherin cluster is driven mainly by lineage-specific gene duplications and degeneration. Our analysis also shows that loss of the protocadherin δ subcluster in the mammalian lineage occurred after the divergence of mammals and reptiles. We present a model for the evolutionary history of the protocadherin cluster in tetrapods.     

The effects of progesterone on sexual behavior in male green anole lizards (Anolis carolinensis)

It is well known that androgen-dependent sexual behaviors in male mammals and birds are inhibited by exogenous progesterone (P). However, recent research on male whiptail lizards (Cnemidophorus inornatus) indicates that P can stimulate sexual and copulatory behavior. We report here both antiandrogenic and synandrogenic actions of P on sexual behavior in males of another reptile, the green anole lizard (Anolis carolinensis). Earlier reports on birds and mammals are reviewed and discussed in relation to a possible physiological role of P in influencing sexual behavior in male vertebrates.     

Sex determination in humans

In mammals, the Y chromosome induces testis formation and thus male sexual development; in the absence of a Y chromosome, gonads differentiate into ovaries and female development ensues. Molecular genetic studies have identified the Y-located testis determining gene SRY as well as autosomal and X-linked genes necessary for gonadal development. The phenotypes resulting from mutation of these genes, together with their patterns of expression, provide the basis for establishing a hierachy of genes and their interactions in the mammalian sex determination pathway.     

Somatic Expression and Autosomal Inheritance of Phosphoglycerate Kinase B in Kangaroos

The PGK-B isozyme, currently known as PGK-2 in the mouse nomenclature, is the predominant PGK isozyme in mammalian sperm. In many species it is detectable only in sperm, in spermatogenic testes and in epididymides containing sperm. In this paper, we provide evidence that some kangaroo species express low PGK-B activity in somatic tissues, in addition to high activity in testes. Three kangaroo species, M. rufogriseus, M. robustus and M. giganteus, exhibit polymorphism of PGK-B. Breeding data support the hypothesis of autosomal co-dominant inheritance, as is the case in mice. Population data for the three polymorphisms are discussed. PGK-B is not detectable in somatic tissues or spermatogenic testis extracts of monotreme mammals, birds or lizards; it is probably restricted to therian mammals.     

Keratin intermediate filament chains in the European common wall lizard (Podarcis muralis) and a potential keratin filament crosslinker

On the basis of sequence homology with mammalian α-keratins, and on the criteria that the coiled-coil segments and central linker in the rod domain of these molecules must have conserved lengths if they are to assemble into viable intermediate filaments, a total of 28 Type I and Type II keratin intermediate filament chains (KIF) have been identified from the genome of the European common wall lizard (Podarcis muralis). Using the same criteria this number may be compared to 33 found here in the green anole lizard (Anole carolinensis) and 25 in the tuatara (Sphenodon punctatus). The Type I and Type II KIF genes in the wall lizard fall in clusters on chromosomes 13 and 2 respectively. Although some differences occur in the terminal domains in the KIF chains of the two lizards and tuatara, the similarities between key indicator residues – cysteine, glycine and proline – are significant. The terminal domains of the KIF chains in the wall lizard also contain sequence repeats commonly based on glycine and large apolar residues and would permit the fine tuning of physical properties when incorporated within the intermediate filaments. The H1 domain in the Type II chain is conserved across the lizards, tuatara and mammals, and has been related to its role in assembly at the 2–4 molecule level. A KIF-like chain (K80) with an extensive tail domain comprised of multiple tandem repeats has been identified as having a potential filament-crosslinking role.     

New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.     

Conservation and function of Dazl in promoting the meiosis of goat male germline stem cells

Dazl (deleted in azoospermia-like) is a conserved gene in mammalian meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Up to date, the expression and function of Dazl in the goat testis are unknown. The objectives of this study were to investigate the expression pattern of Dazl in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Dazl in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. The dairy goat Dazl is highly conserved analysed by several online and bioinformatics software, respectively. Over-expression of Dazl promoted the expression of meiosis-related genes in dairy goat mGSCs. The expression of Stra8 was up-regulated by over-expression of Dazl analysed by Luciferase reporter assay. Taken together, results suggest the Dazl plays an important role in dairy goat spermatogenesis and that over-expression of Dazl may promote Stra8 expression in dairy goat mGSCs.     

Cytological evaluation of spermatogenesis within the germinal epithelium of the male European wall lizard, Podarcis muralis

The annual cytological changes to the male germinal epithelium were investigated in an introduced population of European wall lizards (Podarcis muralis). Testicular tissues were collected, embedded, sectioned by an ultramicrotome, and stained with the PAS procedure followed by a toluidine counterstain. Spermatogenesis in the lizard is divided into the proliferative, meiotic, and maturational phases. Wall lizards have a prenuptial pattern of spermatogenesis, where sperm development begins immediately prior to and continues through the months of breeding (April-June). The testis then involutes, undergoes a short period of quiescence, and recrudescence commences in mid-July. Germ cells undergo proliferation, meiosis, and the early stages of spermiogenesis (maturation) from late July through December. However, the late stages of spermiogenesis are retarded from December through February. Spermiogenesis continues at an accelerated pace from March through May, leading to a single massive spermiation event through the month of June. Although spatial relationships are seen between germ cells within the seminiferous epithelium, accumulation of spermatids during winter and acceleration of elongation in spring prevents determination of consistent cellular associations between early and late developing germ cells within the wall lizard testis. This temporal germ cell development is different from the consistent spatial development seen within seasonally breeding birds and mammals and may represent an evolutionary intermediate in terms of amniotic germ cell development.     

Marsupial anti-Mullerian hormone gene structure,regulatory elements,and expression

During male sexual development in reptiles, birds, and mammals, anti-Müllerian hormone (AMH) induces the regression of the Müllerian ducts that normally form the primordia of the female reproductive tract. Whereas Müllerian duct regression occurs during fetal development in eutherian mammals, in marsupial mammals this process occurs after birth. To investigate AMH in a marsupial, we isolated an orthologue from the tammar wallaby (Macropus eugenii) and characterized its expression in the testes and ovaries during development. The wallaby AMH gene is highly conserved with the eutherian orthologues that have been studied, particularly within the encoded C-terminal mature domain. The N-terminus of marsupial AMH is divergent and larger than that of eutherian species. It is located on chromosome 3/4, consistent with its autosomal localization in other species. The wallaby 5' regulatory region, like eutherian AMH genes, contains binding sites for SF1, SOX9, and GATA factors but also contains a putative SRY-binding site. AMH expression in the developing testis begins at the time of seminiferous cord formation at 2 days post partum, and Müllerian duct regression begins shortly afterward. In the developing testis, AMH is localized in the cytoplasm of the Sertoli cells but is lost by adulthood. In the developing ovary, there is no detectable AMH expression, but in adults it is produced by the granulosa cells of primary and secondary follicles. It is not detectable in atretic follicles. Collectively, these studies suggest that AMH expression has been conserved during mammalian evolution and is intimately linked to upstream sex determination mechanisms.     

Analysis of Transient Receptor Potential Ankyrin 1 (TRPA1) in Frogs and Lizards Illuminates Both Nociceptive Heat and Chemical Sensitivities and Coexpression with TRP Vanilloid 1 (TRPV1) in Ancestral Vertebrates

Transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (V1) perceive noxious temperatures and chemical stimuli and are involved in pain sensation in mammals. Thus, these two channels provide a model for understanding how different genes with similar biological roles may influence the function of one another during the course of evolution. However, the temperature sensitivity of TRPA1 in ancestral vertebrates and its evolutionary path are unknown as its temperature sensitivities vary among different vertebrate species. To elucidate the functional evolution of TRPA1, TRPA1s of the western clawed (WC) frogs and green anole lizards were characterized. WC frog TRPA1 was activated by heat and noxious chemicals that activate mammalian TRPA1. These stimuli also activated native sensory neurons and elicited nocifensive behaviors in WC frogs. Similar to mammals, TRPA1 was functionally co-expressed with TRPV1, another heat- and chemical-sensitive nociceptive receptor, in native sensory neurons of the WC frog. Green anole TRPA1 was also activated by heat and noxious chemical stimulation. These results suggest that TRPA1 was likely a noxious heat and chemical receptor and co-expressed with TRPV1 in the nociceptive sensory neurons of ancestral vertebrates. Conservation of TRPV1 heat sensitivity throughout vertebrate evolution could have changed functional constraints on TRPA1 and influenced the functional evolution of TRPA1 regarding temperature sensitivity, whereas conserving its noxious chemical sensitivity. In addition, our results also demonstrated that two mammalian TRPA1 inhibitors elicited different effect on the TRPA1s of WC frogs and green anoles, which can be utilized to clarify the structural bases for inhibition of TRPA1.     

Proteomic analysis of male 4C germ cell proteins involved in mouse meiosis

Male meiosis is a specialized type of cell division that gives rise to sperm. Errors in this process can result in the generation of aneuploid gametes, which are associated with birth defects and infertility in humans. Until now, there has been a lack of a large-scale identification of proteins involved in male meiosis in mammals. In this study, we report the high-confidence identification of 3625 proteins in mouse male germ cells with 4C DNA content undergoing meiosis I. Of these, 397 were found to be testis specific. Bioinformatics analysis of the proteome led to the identification of 28 proteins known to be essential for male meiosis in mice. We also found 172 proteins that had yeast orthologs known to be essential for meiosis. Chromosome distribution analysis of the proteome showed underrepresentation of the identified proteins on the X chromosome, which may be due to meiotic sex chromosome inactivation. Characterization of the proteome of 4C germ cells from mouse testis provides an inventory of proteins, which is useful for understanding meiosis and the mechanisms of male infertility.     

Expression pattern of Boule in dairy goat testis and its function in promoting the meiosis in male germline stem cells (mGSCs)

Boule is a conserved gene in meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Deletion of Boule was found to block meiosis in spermatogenesis, which contributes to infertility. Up to date, the expression and function of Boule in the goat testis are not known. The objectives of this study were to investigate the expression pattern of Boule in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Boule in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. Over‐expression of Boule promoted the expression of meiosis‐related genes in dairy goat mGSCs. The expression of Stra8 was up‐regulated by over‐expression of Boule analyzed by Western blotting and Luciferase reporter assay. While, Cdc25a, the downstream regulator of Boule, was found not to affect the expression of Stra8, and our data illustrated that Cdc25a did not regulate meiosis via Stra8. The expression of Stra8 and Boule was up‐regulated by RA induction. Taken together, results suggest the Boule plays an important role in dairy goat spermatogenesis and that over‐expression of Boule may promote spermatogenesis and meiosis in dairy goat. J. Cell. Biochem. 114: 294–302, 2013. © 2012 Wiley Periodicals, Inc.     

Sexually dimorphic estrogen receptor α mRNA expression in the preoptic area and ventromedial hypothalamus of green anole lizards

Estradiol (E2) is important in activation of male reproductive behaviors, and masculinizes morphology of associated brain regions in a number of mammalian and avian species. In contrast, it is testosterone, rather than its metabolites, that is the most potent activator of male sexual behavior in green anole lizards. As in other vertebrate groups, however, E2 is critical for receptivity in females of this species. Aromatase, the enzyme which converts testosterone to E2, is more active in the male than female green anole brain, and appears to be actively regulated on a seasonal basis, suggesting some role for E2 in males. This study was designed to enhance our understanding of potential E2 actions by localizing and quantifying relative levels of estrogen receptor-alpha (ERα) mRNA in forebrain regions involved in masculine and feminine behaviors in anoles. These areas include the preoptic area (POA), ventromedial amygdala (AMY) and ventromedial hypothalamus (VMH). In situ hybridization was conducted in adult males and females collected during both breeding and non-breeding seasons. ERα mRNA was expressed in each brain region across sexes and seasons. However, expression was up to 3 times greater in the VMH compared to the POA and AMY. In the POA and VMH, expression was higher in females compared to males, independent of season. The increased receptor expression in females is consistent with E2 playing a larger role in female than male reproductive behaviors.     

Pattern and density of vascularization in mammalian testes, ovaries, and ovotestes

According to the classical paradigm, the vasculature of the embryonic testis is more dense and complex than that of the ovary, but recent studies based on whole-mount detection of Caveolin-1 (CAV1) as an endothelial cell marker, have suggested that the level of ovarian vascularization is higher than previously assumed. However, this new hypothesis has been neither tested using alternative methodology nor investigated in other mammalian species. In this paper, we have studied the vascularization process in the gonads of males and females of two mammalian species, the mouse (Mus musculus) and the Iberian mole (Talpa occidentalis). Our results show that the pattern of testis vascularization is very well conserved among mammals, including both pre- and postnatal stages of development and, at least in the mole, it is conserved irrespectively of whether the testicular tissue is XY or XX. We have shown that CAV1 is present not only in endothelial cells but also in prefollicular oocytes and in an ovarian population of somatic cortical cells. These data clearly establish that: (1) according to the classical hypothesis, the degree of vascularization of the developing ovary is lower than that of the testis, (2) ovarian vascularization is also evolutionarily conserved as it occurs similarly both in moles and in mice, and (3) that the degree of vascular development of the mammalian ovary is age-dependent increasing significatively at puberty. The expression of CAV1 in the ovary of most animal taxa, from nematodes to mammals, strongly suggests a role for this gene in the female meiosis.