Paweł Grzmil

The Small Heat Shock Protein ODF1/HSPB10 Is Essential for Tight Linkage of Sperm Head to Tail and Male Fertility in Mice

ABSTRACT Sperm motility and hence male fertility strictly depends on proper development of the sperm tail and its tight anchorage to the head. The main protein of sperm tail outer dense fibers, ODF1/HSPB10, belongs to the family of small heat shock proteins that function as molecular chaperones. However, the impact of ODF1 on sperm tail formation and motility and on male fecundity is unknown. We therefore generated mutant mice in which the Odf1 gene was disrupted. Heterozygous mutant male mice are fertile while sperm motility is reduced, but Odf1-deficient male mice are infertile due to the detachment of the sperm head. Although headless tails are somehow motile, transmission electron microscopy revealed disturbed organization of the mitochondrial sheath, as well as of the outer dense fibers. Our results thus suggest that ODF1, besides being involved in the correct arrangement of mitochondrial sheath and outer dense fibers, is essential for rigid junction of sperm head and tail. Loss of function of ODF1, therefore, might account for some of the cases of human infertility with decapitated sperm heads. In addition, since sperm motility is already affected in heterozygous mice, impairment of ODF1 might even account for some cases of reduced fertility in male patients.

Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA, and inflammatory factors

Abstract The caseinolytic peptidase P (CLPP) is conserved from bacteria to humans. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder together with the chaperone CLPX. In spite of a known relevance for the mitochondrial unfolded protein response, its substrates and tissue-specific roles are unclear in mammals. Recessive CLPP mutations were recently observed in the human Perrault variant of ovarian failure and sensorineural hearing loss. Here, a first characterization of CLPP null mice demonstrated complete female and male infertility and auditory deficits. Disrupted spermatogenesis already at the spermatid stage and ovarian follicular differentiation failure were evident. Reduced pre-/post-natal survival and marked ubiquitous growth retardation contrasted with only light impairment of movement and respiratory activities. Interestingly, the mice showed resistance to ulcerative dermatitis. Systematic expression studies detected up-regulation of other mitochondrial chaperones, accumulation of CLPX and mtDNA as well as inflammatory factors throughout tissues. T-lymphocytes in the spleen were activated. Thus, murine Clpp deletion represents a faithful Perrault model. The disease mechanism probably involves deficient clearance of mitochondrial components and inflammatory tissue destruction.

Human cyritestin genes (CYRN1 and CYRN2) are non-functional.

The mouse cyritestin gene is a member of the ADAM (a disintegrin and metalloprotease) gene family and codes for a membrane-anchored sperm protein. Recently, it was shown that cyritestin is critical for male fertility in the mouse. Spermatozoa of cyritestin-deficient mice are not able to bind to the zona pellucida of the oocyte and therefore unable to fertilize the egg. However, zona-free oocytes can be fertilized and the resulting embryos show normal development. In contrast to the mouse, where only one gene for cyritestin (Cyrn) is reported, two cyritestin genes (CYRN1 and CYRN2) are known in humans. The human CYRN1 and CYRN2 genes are located on chromosomes 8 and 16, respectively. We report that 27% of fertile men are deficient for the CYRN1 gene but that all have a CYRN2 gene, suggesting that the CYRN2 gene is the orthologous mouse cyritestin gene in humans and might be involved in sperm-egg interactions. However, the characterization of CYRN2 transcripts from testicular RNA of CYRN1-deficient men demonstrated many termination codons in the synthesized cyritestin cDNA. Furthermore, Western-blot analysis with human testicular protein extracts using an anti-cyritestin antibody failed to detect any cyritestin protein. These results demonstrate clearly that both cyritestin genes are non-functional in humans.

Inactivation of Insulin-Like Factor 6 Disrupts the Progression of Spermatogenesis at Late Meiotic Prophase

Insulin-like factor 6 (INSL6), a member of the insulin-like superfamily, is predominantly expressed in male germ cells. Expression of the Insl6 is first detected in mouse testis at postnatal d 15 when the first wave of spermatogenesis progresses to pachytene spermatocytes. To elucidate the role of INSL6 in germ cell development, we generated Insl6-deficient mice. The majority of the Insl6-deficient males on a hybrid genetic background exhibited impaired fertility, whereas females were fertile. The number of mature sperm and sperm motility were drastically reduced in the epididymis. The reduced sperm count could be due to apoptotic death of a significant number of developing germ cells. Analysis of germ cell development during the juvenile life showed an arrest of the first wave of spermatogenesis in late meiotic prophase. RNA analysis revealed a significant decrease in expression of late meiotic- and postmeiotic-specific marker genes, whereas expression of early meiotic-specific genes remains unaffected in the Insl6(-/-) testes. These results demonstrate that INSL6 is required for the progression of spermatogenesis.

Grossuana codreanui (Grossu, 1946) and the phylogenetic relationships of the East Balkan genus Grossuana (Radoman, 1973) (Gastropoda: Rissooidea)

The rich rissooidean fauna of Balkan Peninsula, the history of which began at the time of Paratethys, is not sufficiently studied yet. It includes, among others, many taxa of minute gastropods with a moderately high-spired shell, a penis with a double lobe on its left edge and female reproductive organs with a loop of coiled oviduct and two receptacles, the morphology of which does not allow for a certain determination. In the article we deal with five such populations. We describe the shell, radula, soft part morphology and anatomy of the reproductive organs of Grossuana codreanui (Grossu, 1946) from its type locality at the Techirghiol Lake, Dobrogea, Romania. The species is compared molecularly (fragments of two genes: mitochondrial CO1 and ribosomal 18 S) with G. codreanui from Bulgaria, G. serbica (Radoman, 1973) from its type locality, Belgrandiella haesitans (Westerlund, 1881) from the spring of the Louros River, and Orientalina delphica (Radoman, 1973) from the spring Kastalia at Delphi. All the five populations were found to belong to the genus Grossuana: G. codreanui, G. serbica, G. vurliana (Radoman, 1966), and G. dephica (Radoman, 1973), respectively. We applied 12 morphological characters of Grossuana and 17 other rissooidean genera (mostly of the Hydrobiidae s. stricto) to infer phylogeny, which has shown the position of the genus within the Hydrobiidae s. stricto, most close to Orientalina. All the molecular data confirm that Grossuana is a distinct genus belonging to the Hydrobiidae, Horatiinae, whose closest relatives are Belgrandia, Orientalina and Horatia.

Prm3, the Fourth Gene in the Mouse Protamine Gene Cluster, Encodes a Conserved Acidic Protein That Affects Sperm Motility

The protamine gene cluster containing the Prm1, Prm2, Prm3, and Tnp2 genes is present in humans, mice, and rats. The Prm1, Prm2, and Tnp2 genes have been extensively studied, but almost nothing is known about the function and regulation of the Prm3 gene. Here we demonstrate that an intronless Prm3 gene encoding a distinctive small acidic protein is present in 13 species from seven orders of mammals. We also demonstrate that the Prm3 gene has not generated retroposons, which supports the contention that genes that are expressed in meiotic and haploid spermatogenic cells do not generate retroposons. The Prm3 mRNA is first detected in early round spermatids, while the PRM3 protein is first detected in late spermatids. Thus, translation of the Prm3 mRNA is developmentally delayed similar to the Prm1, Prm2, and Tnp2 mRNAs. In contrast to PRM1, PRM2, and TNP2, PRM3 is an acidic protein that is localized in the cytoplasm of elongated spermatids and transfected NIH-3T3 cells. To elucidate the function of PRM3, the Prm3 gene was disrupted by homologous recombination. Sperm from Prm3(-/-) males exhibited reductions in motility, but the fertility of Prm3(-/-) and Prm3(+/+) males was similar in matings of one male and one female. We have developed a competition test in which a mutant male has to compete with a rival wild-type male to fertilize a female; the implications of these results are also discussed.

Overexpression of peroxisomal testis-specific 1 protein induces germ cell apoptosis and leads to infertility in male mice.

The overexpression of PXT1 in mouse testis leads to germ cell apoptosis and male infertility. The PXT1 protein contains a functional BH3-motif. PXT1 protein interacts with BAT3, and BAT3 is able to protect cells from PXT1-induced apoptosis.

Disruption of the murine dynein light chain gene Tcte3-3 results in asthenozoospermia

To elucidate the role of the mouse gene Tcte3 (Tctex2), which encodes a putative light chain of the outer dynein arm of cilia and sperm flagella, we have inactivated this gene in mice using targeted disruption. Breeding of heterozygous males and females resulted in normal litter size; however, we were not able to detect homozygous Tcte3-deficent mice using standard genotype techniques. In fact, our results indicate the presence of at least three highly similar copies of the Tcte3 gene (Tcte3-1, Tcte3-2, and Tcte3-3) in the murine genome. Therefore, quantitative real-time PCR was established to differentiate between mice having one or two targeted Tcte3-3 alleles. By this approach, Tcte3-3(-/-) animals were identified, which were viable and revealed no obvious malformation. Interestingly, some homozygous Tcte3-3-deficient male mice bred with wild-type female produced no offspring while other Tcte3-3-deficient males revealed decreased sperm motility but were fertile. In infertile Tcte3-3(-/-) males, spermatogenesis was affected and sperm motility was reduced, too, resulting in decreased ability of Tcte3-3-deficient spermatozoa to move from the uterus into the oviduct. Impaired flagellar motility is not correlated with any gross defects in the axonemal structure, since outer dynein arms are detectable in sperm of Tcte3-3(-/-) males. However, in infertile males, deficient Tcte3-3 function is correlated with increased apoptosis during male germ cell development, resulting in a reduction of sperm number. Moreover, multiple malformations in developing haploid germ cells are present. Our results support a role of Tcte3-3 in generation of sperm motility as well as in male germ cell differentiation.

Daphniola Radoman, 1973 (Gastropoda: Hydrobiidae): shell biometry, mtDNA, and the Pliocene flooding

Shell biometry and cytochrome c oxidase subunit 1 (CO1) mtDNA were studied in Daphniola exigua (Schmidt, 1856), D. graeca Radoman, 1973, and D. louisi Falniowski and Szarowska, 2000 from Greece. Principal component analysis of shell morphometry confirmed the distinctness of D. louisi along the PC3 axis. Kimura 2‐parameter (K2P) genetic distances within D. graeca and D. exigua were 0.016 and 0.003–0.008, respectively, all D. louisi sequences were identical. The distance between D. exigua and D. graeca was 0.013–0.027. The distances between D. louisi and D. graeca, and D. louisi and D. exigua, were 0.098–0.110 and 0.091–0.096, respectively. The mean distance between D. louisi and the other Daphniola species was 0.098±0.007, while between the eight Daphniola specimens and the so far closest species Grossuana codreanui (Grossu, 1946) was 0.102–0.123. A maximum likelihood tree was constructed for all Daphniola, with Grossuana codreanui and Bythinella austriaca (Frauenfeld, 1856) as an outgroup. This confirmed that D. louisi is a distinct species, and must have diverged after the Pliocene marine transgression.

PHF5A represents a bridge protein between splicing proteins and ATP-dependent helicases and is differentially expressed during mouse spermatogenesis

PHF5A is a highly conserved protein from yeast to man, and based on studies in yeast, it was suggested that the homologous protein RDS3P in S. cerevisiae takes part in the organization of U2 snRNP particles. By using the yeast two-hybrid assay we could demonstrate that PHF5A interacted both with ATP-dependent helicases EP400 and DDX1 and with arginine-serine (RS)-rich domains of splicing factors U2AF1 and SFRS5 in mouse. Furthermore, domain interaction studies revealed that PHF5A interaction with EP400 and DDX1 is restricted to the N-terminal part of PHF5A, whereas the C-terminal region of PHF5A was found to be responsible for the association with U2AF1 and SFRS5. By using the yeast three-hybrid assay, we could further show that both EP400 and DDX1 interacted only indirectly with U2AF1 and SFRS5 proteins via the bridge protein PHF5A. The subcellular localization of a PHF5A-GFP fusion protein was predominantly observed in the nucleus and, in addition, PHF5A co-localized with both U2AF1 and SFRS5 proteins in nuclear speckles of NIH3T3 cells. Moreover, expression analyses demonstrated that PHF5A and U2AF1 gene expression coincided in spermatocytes during murine spermatogenesis and interaction between these proteins was also detectable in the spermatocyte-specific cell line GC-4spc by using in vivo co-immunoprecipitation studies. Taken together, our results indicate that PHF5A resembles a protein which interacts with splicing factors U2AF1 and SFRS5 and helicases EP400 and DDX1 and functions as a bridge protein between these proteins.