The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.Sperm chromatin condensation during the final steps of spermatogenesis in mammals is a multistep process that includes the sequential replacement of the majority of histones by transition proteins and protamines in testis (6, 7). During epididymal transit of spermatozoa, protamine thiol oxidation is completed and intra-and intermolecular cross-links are formed. Hence, a transcriptionally inactive and tightly packed haploid genome is generated rendering sperm nuclei more resistant to mechanical and chemical insults (2). Recently, Cho and colleagues showed that chimeric mice hemizygous for protamine 1 or 2 fail to transmit the targeted allele to the germ line (8).Selenium depletion studies of rodents clearly demonstrated the importance of this trace element in male fertility. Third generation selenium deficiency is associated with structural abnormalities, such as broken midpieces of sperm tails, giant heads, and reversible testicular atrophy (5, 34). Due to its particular high expression in mammalian testis (21) and its resistance to selenium deprivation in testis, the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is thought to account for most of the defects associated with severe selenium deficiency.PHGPx was initially characterized as a lipid peroxidationinhibiting protein (33) and was later shown to be an unusual member of the glutathione peroxidase family, in particular for its scarce specificity for both the oxidizing and reducing substrates (32). Most relevant in this respect was the observation that, in the presence of low glutathione (GSH) concentration, specific protein -SH groups may act as a reductant in the catalytic cycle with a stoichiometry of 2 equivalents of thiol per mole of hydroperoxide (13,22,...

Genetic variants underlying reduced male reproductive performance have been identified in humans and model organisms, most of them compromising semen quality. Occasionally, male fertility is severely compromised although semen analysis remains without any apparent pathological findings (i.e., idiopathic subfertility). Artificial insemination (AI) in most cattle populations requires close examination of all ejaculates before insemination. Although anomalous ejaculates are rejected, insemination success varies considerably among AI bulls. In an attempt to identify genetic causes of such variation, we undertook a genome-wide association study (GWAS). Imputed genotypes of 652,856 SNPs were available for 7962 AI bulls of the Fleckvieh (FV) population. Male reproductive ability (MRA) was assessed based on 15.3 million artificial inseminations. The GWAS uncovered a strong association signal on bovine chromosome 19 (P = 4.08×10−59). Subsequent autozygosity mapping revealed a common 1386 kb segment of extended homozygosity in 40 bulls with exceptionally poor reproductive performance. Only 1.7% of 35,671 inseminations with semen samples of those bulls were successful. None of the bulls with normal reproductive performance was homozygous, indicating recessive inheritance. Exploiting whole-genome re-sequencing data of 43 animals revealed a candidate causal nonsense mutation (rs378652941, c.483C>A, p.Cys161X) in the transmembrane protein 95 encoding gene TMEM95 which was subsequently validated in 1990 AI bulls. Immunohistochemical investigations evidenced that TMEM95 is located at the surface of spermatozoa of fertile animals whereas it is absent in spermatozoa of subfertile animals. These findings imply that integrity of TMEM95 is required for an undisturbed fertilisation. Our results demonstrate that deficiency of TMEM95 severely compromises male reproductive performance in cattle and reveal for the first time a phenotypic effect associated with genomic variation in TMEM95.

Using a digital videomicroscopic analysis system in the bovine, we showed that the mechanisms of transport caused by ciliary beating are distinctly different in ampulla and isthmus of the oviduct. The average particle transport speed (PTS) in the oviduct (mean, 133 microm/sec) does not differ in the cycle (metestrus) and during pregnancy after implantation, but it is locally modulated at the site of the embryo. Using videomicroscopy, we were able to document that after entering the ampulla, the cumulus-oocyte complex (COC) is not transported by ciliary beating down the oviduct, but firmly attaches to the ampullar epithelium. This attachment is mediated by the cumulus cells. However, when a COC is degenerated, it is floating in the oviductal lumen. As soon as a vital COC is in the ampulla, the sperm bound in the sperm reservoir of the ampullar isthmic junction leave the reservoir and hurry to the oocyte. When a sperm has penetrated the zona pellucida, the COC detaches and continues its migration. Quantitative measurements showed that the early embryo is able to locally downregulate PTS during its migration down the oviduct. It locally changes the pattern of vascularization and induces the formation of secretory cells. Our studies imply that the oviductal epithelium is able to select vital oocytes. The early embryo is able to induce the formation of secretory cells, modify vascularization, and downregulate speed of transport, thus creating the prerequisite for the first embryo-maternal communication in the oviduct.

Early embryonic development, implantation and maintenance of a pregnancy are critically dependent on an intact embryo-maternal communication. So far, only few signals involved in this dialogue have been identified. In bovine and other ruminants, interferon tau is the predominant embryonic pregnancy recognition signal, exhibiting antiluteolytic activity. However, this is just one aspect of the complex process of embryo-maternal signalling, and a number of other systems are more likely to be involved. To gain a more comprehensive understanding of these important mechanisms, integrated projects involving specialists in embryology, reproductive biotechnology and functional genome research are necessary to perform a systematic analysis of interactions between pre-implantation stage embryos and oviduct or uterine epithelial cells, respectively. State-of-the-art transcriptomic and proteomic technologies will identify reciprocal signals between embryos and their maternal environment and the respective downstream reaction cascades. For in vivo studies, the use of monozygotic twins as recipient animals provides elegant model systems, thus eliminating genetic variability as a cause of differential gene expression. In addition, suitable systems for the co-culture of oviduct epithelial or endometrium cells with the respective embryonic stages need to be established for functional validation of candidate genes potentially involved in the dialogue between embryos and their maternal environment. The knowledge of these mechanisms should help to increase the pregnancy rate following embryo transfer and to avoid embryonic losses. Candidate genes involved in embryo-maternal communication will also be used to define new quality criteria for the selection of embryos for transfer to recipients. Another application is the supplementation of embryotrophic factors or components of embryo-maternal signalling in optimized formulations, such as bioartificial matrices. As a long-term goal, signalling mechanisms identified in bovine will also be functionally evaluated in other species, including the human.

By reverse transcription-polymerase chain reaction, the transcript of the growth hormone receptor (GHR) was demonstrated in oocytes, follicular cells, and corpus luteum of the bovine ovary. Immunoblotting using the monoclonal antibody mAb 263 resulted in a distinct protein band at 120 kDa, confirming that translation of the mRNA takes place in the same cells. Nonradioactive in situ hybridization revealed that distribution of the mRNA encoding GHR was correlated with the developmental stage of the follicle. Whereas in primordial and primary follicles the oocyte showed distinct amounts of the transcript encoding GHR, in tertiary follicles the mRNA was predominantly localized in the cells of the cumulus oophorus. GHR mRNA was also expressed in the large granulosa lutein cells, in the germinal epithelium, and in the endothelial cells of ovarian vessels. Colocalization of the GHR protein showed a distribution pattern identical to that of the mRNA. In calves, oocyte and follicle cells changed GHR expression in the same way as in the adult ovary. During embryonic development of the ovary, distinct amounts of the mRNA encoding GHR were found in primordial follicles shortly before birth. Our results imply that the GHR is involved in ovarian ontogenesis, especially in early folliculogenesis.

No abstract

TFF-peptides (formerly P-domain peptides, trefoil factors) represent major secretory products of the mammalian gastrointestinal tract. A molecular cloning approach revealed the existence of two TFF-peptides, xP1 and xP4, also in the stomach of Xenopus laevis. Here, the localization of these two peptides by Western blot analysis as well as immunohistochemistry is presented. xP1 is found predominantly in the surface mucous cells of the stomach, whereas xP4 is mainly localized to a specific population of goblet cells in the esophagus, to mucous neck cells of the stomach, and to closely resembling cells in antral glands. xP4 in the esophagus and in the stomach differ by their N-glycosylation patterns. Compared to mammalian TFF-peptides, xP1 obviously represents the frog homologue of human TFF1 (formerly pS2) and xP4 seems to be the amphibian equivalent of human TFF2 (formerly hSP).

Elevated estradiol levels are correlated with male infertility. Causes of hyperestrogenism include diseases of the adrenal cortex, testis or medications affecting the hypothalamus-pituitary-gonadal axis. The aim of our study was to elucidate the effects of estradiol treatment on testicular cellular morphology and function, with reference to the treatment regimen received. Testes samples (n = 9) were obtained post-orchiectomy from male-to-female transsexuals within the age range of 26–52 years. Each patient had a minimum of 1–6 years estradiol treatment. For comparison, additional samples were obtained from microscopically unaltered testicular tissue surrounding tumors (n = 7). The tissues obtained were investigated by stereomicroscopy, histochemistry, scanning electron microscopy (SEM) and immunohistochemistry. Our studies revealed that estradiol treatment significantly decreased the diameter of the seminiferous tubules (p < 0.05) and induced fatty degeneration in the surrounding connective tissue. An increase in collagen fiber synthesis in the extracellular matrix (ECM) surrounding the seminiferous tubules was also induced. Spermatogenesis was impaired resulting in mainly spermatogonia being present. Sertoli cells revealed diminished expression of estrogen receptor alpha (ERα). Both Sertoli and Leydig cells showed morphological alterations and glycoprotein accumulations. These results demonstrate that increased estradiol levels drastically impact the human testis.