Although the main theme of this volume includes the composition of human milk and the specific requirements for different components of milk for infant nutrition, our interest in milk proteins has been generated for rather different reasons. For a number of years we have been interested in the many complex intracellular mechanisms involved in the expression of structural genes in higher organisms. The lactating mammary gland provides an attractive system for a variety of reasons. Early studies based on antibody precipitation procedures using antibodies raised against mainly rat, rabbit, or mouse milk proteins demonstrated that milk protein gene expression was modulated by an intricate combination of steroid and peptide hormones (1), although the precise intracellular mechanism of action of these hormones remained to be established. With the development of techniques for the rapid isolation of eucaryote mRNA and the identification of mRNA species by cell-free protein synthesis, interest in the intracellular mechanisms involved in the synthesis of mammalian proteins increased. Invariably, studies were based on those systems in which a few well-defined proteins were produced in response to specific hormonal stimuli. Consequently, as the milk proteins fall into this category, the manner in which they are synthesized and secreted by the cells of the mammary gland has important implications for biology far beyond their basic relevance to lactation.

A number of MDC (metalloproteinase-like, disintegrin-like, cysteine-rich) proteins are exclusively or abundantly expressed on mammalian sperm from a range of species, with data from rodents supporting a role for some of these in oolemma binding. However, in the human, transcripts for three of the most likely candidates have been shown to contain deletions and in-frame termination codons, rendering them non-functional. In this paper we have addressed the expression, in humans, of an additional MDC protein, tMDC III (also known as ADAM 18), previously shown to be exclusively expressed on sperm from rodents and macaques. Using a PCR-based approach, we have determined the complete nucleotide sequence of human tMDC III cDNA which, unlike the three non-functional genes, contains an uninterrupted open reading frame encoding a full-length MDC protein. Furthermore, polyclonal antisera raised against human recombinant tMDC III demonstrated the presence of mature protein on human sperm. In common with orthologues from other species, human tMDC III contains a putative integrin-binding glu-cys-asp (ECD) motif, the relevance of which is discussed in the context of a possible role in oolemma binding.

Sp17 was originally proposed to be a sperm-specific protein and thought to play a role in sperm-egg interactions by binding to the zona pellucida via two conserved heparin-binding motifs. However, more recent data indicate that it may be expressed more widely, both in tumours and in normal somatic tissues. The complete nucleotide and deduced amino acid sequences of human Sp17 transcripts are reported and it is shown that they are expressed in a wide range of tissues, albeit at a much lower expression than in the testis. On the basis of the extremely high sequence conservation throughout the N-terminal half of Sp17, and the presence within this region of an A-kinase anchoring protein (AKAP)-binding motif, it is postulated that the proposed role of Sp17 in zona pellucida binding is unlikely to be its principal function.

Integrins have been proposed to play a role in mammalian sperm-oocyte interactions for many years. To a large extent this hypothesis stems from the ability of short synthetic peptides, based on the disintegrin-like domains of two sperm surface integral membrane proteins, fertilin beta and cyritestin, to inhibit sperm--oocyte binding and fusion in vitro. Here we argue that such peptide mimics lack specificity in these simple IVF assay systems. Hence, whilst not precluding a role for fertilin beta and cyritestin in sperm-oolemma interactions, this lack of specificity indicates the need for considerable caution when interpreting results obtained using this approach.

Rat sperm 2B1 antigen (the orthologue of guinea pig sperm PH20) is a plasma membrane-bound glycoprotein that is endoproteolytically cleaved during passage through the epididymis and subsequently migrates from the tail to the acrosomal domain during capacitation. Unlike guinea pig PH20, however, sperm surface 2B1 is insensitive to phosphatidylinositol phospholipase C, nor is it known how endoproteolytic cleavage affects its hyaluronidase activity. In this investigation we have expressed 2B1 cDNA in Chinese hamster ovary cells; we have shown that it contains an internal sequence motif for attachment of a glycosyl phosphatidylinositol (GPI) anchor and that cleavage from a single- into a two-chain molecule causes a significant shift in the optimum pH for hyaluronidase activity. Functionally, these results suggest that 1) 2B1 glycoprotein on rat spermatozoa is attached to the plasma membrane via a GPI anchor and that this is an important factor in its ability to migrate from the tail to the acrosomal domain during capacitation; and 2) endoproteolytic cleavage of 2B1 serves to optimize its hyaluronidase activity immediately before fertilization, thereby facilitating penetration of spermatozoa through the cumulus oophorus.

Objective: To further elucidate cortisol metabolism in the follicular microenvironment at the time of oocyte retrieval, the presence of 11 beta-hydroxysteroid dehydrogenase (HSD) messenger (m)RNA transcripts in oocytes; cumulus cells; granulosa cells; and CD45(+), CD15(+) leukocytes was assessed semiquantitatively.Design: Controlled study using semiquantitative assessment of 11 beta-HSD mRNA.Setting: University IVF center.Patient(s): Twenty-six patients undergoing controlled ovarian hyperstimulation for assisted conception.Intervention(s): Metaphase II oocytes; cumulus cells; granulosa cells, and CD45(+), CD15(+) leukocytes from individual follicular fluid aspirates.Main Outcome Measures: Semiquantitative analysis of PCR products after total RNA extraction and complementary DNA synthesis.Result(s): Periovulatory human oocytes; cumulus cells; CD45(+), CD15(+) leukocytes; and granulosa cells consistently express type 1 but not type 11 beta-HSD mRNA. Expression of mRNA is greatest in cumulus cells. Type 1 11 beta-HSD mRNA expression varies considerably in all cell types and among individual follicles and patients.Conclusion(s): These studies of mRNA expression suggest that the enzymes present both in and around the periovulatory oocyte will favor a high-cortisol environment. (C) 2000 by American Society for Reproductive Medicine.

A feline splenic cDNA library was screened with a (32)P-labelled cDNA probe encoding the canine IgE epsilon heavy chain subunit. A cDNA sequence of 1614 nucleotides encoding the complete feline IgE heavy chain, as well as a portion of a variable region, was identified. A search of the GenBank database revealed an identity of 82% at the nucleotide level and 76% at the amino acid level between the feline epsilon heavy chain sequence and the canine homologue. In a separate study, feline genomic DNA, isolated from whole feline embryo cells, was subjected to PCR amplification using primers based on known partial genomic DNA sequences for the feline C epsilon gene. Following removal of an intron from the 683 bp PCR product, the coding sequence yielded an ORF of 506 bp. The DNA sequence of this PCR clone differed by a single nucleotide from the cDNA clone. This difference is silent, and therefore the proteins encoded by the two sequences are identical over the regions cloned and sequenced. Phylogenetic analysis of the constant regions of nine immunoglobulin epsilon genes revealed that the feline cDNA is most similar to the canine homologue.

Phosphatidylethanolamine-binding protein (PEBP) is a highly-conserved 21- to 23-kDa basic protein that shows preferential affinity in vitro for phosphatidylethanolamine. Previous studies have focussed on PEBP in the brain and male reproductive tract where it has been proposed to play a role in membrane biogenesis. In the present more comprehensive study, rat PEBP transcripts and protein have been found to be expressed in all tissues examined, although the levels vary considerably between tissues. However, at the cellular level, PEBP expression is enigmatic, being restricted to a diverse range of highly specialised neuronal and non-neuronal cell types. The nature of this diversity, ranging from oligodendrocytes to plasma cells, whilst not precluding a role for PEBP in membrane biogenesis in some cell types, would imply that this is not the major function in others.

The advent of simple in vitro fertilisation techniques has provided the reproductive biologist with an invaluable system for assaying sperm fertilising ability. In particular, they provide a useful way of identifying and characterising gamete-specific proteins that play a role in sperm-egg interactions, and in recent years, a growing number of sperm surface proteins have been identified that appear to be involved in these processes. Fertilin beta was one of the first sperm membrane proteins to be implicated in egg interactions and it has been proposed that this is mediated by means of binding of its disintegrin-like domain to cognate integrin receptors on the egg plasma membrane. A recent paper in Science by Cho and colleagues [Cho et al. 1998. Fertilisation defects in sperm from mice lacking fertilin beta. Science 281: 1857-1859 (Ref. 1)] provides preliminary data on a fertilin beta knockout mouse. Whilst fertilin beta null males had greatly reduced fertility, somewhat surprisingly, this could be largely attributed to causes other than impaired binding to the egg plasma membrane.

Newly synthesized mammalian spermatozoa undergo critical modifications as they pass along the epididymis. The modifications endow spermatozoa with fertilizing ability and occur largely as a consequence of epididymal gene expression. With this in mind, we here employed a cDNA cloning strategy designed to identify key epididymal gene products. We describe a novel cynomolgus monkey (Macaca fascicularis) epididymal transcript designated cy-ESP13.2, of 690 nucleotides. The putative human ortholog was cloned and is highly conserved. Both cDNA sequences predict small, secretory proteins with a disulfide-stabilized core. Anti-peptide polyclonal antibodies were raised to a predicted cy-ESP13.2 surface loop. Western blotting with these antibodies revealed high-level, epididymis-specific expression of cy-ESP13.2, consistent with the pattern of cy-ESP13.2 mRNA expression assessed by Northern blotting. cy-ESP13.2 protein was of 30 kDa and was readily detectable in epithelial cells lining the efferent ductules, initial segment, and cauda regions of the epididymis, but not on spermatozoa. Similarities to members of the four-disulfide-core family suggest clues to ESP13.2 function.

Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm egg binding and fertilisation, offers an attractive approach to the growing global problem of overpopulation. Such an idea is not new; indeed several immunocontraception trials, using animal model systems, have been reported in recent years and a number are reviewed here. However, the results of these studies have been largely disappointing. We believe that two fundamental flaws attribute to the poor success of most of these preliminary immunocontraceptive trials. Firstly, loss of fertility has invariably been used as the assay. This presupposes that immuno-neutralisation of a single, gamete-specific antigen will be sufficient to cause a significant reduction in fertility; however, recent data suggests that such a premise may not be well-founded for a number of reasons. Secondly, and arguably the most important flaw, is the almost universal, but largely inappropriate, use of systemic immunisation as the sole route of antigen delivery. Whilst systemic immunisation regimes may lead to high serum IgG levels, these levels do not correlate with specific antibody levels in the reproductive tract or with contraceptive efficacy. Hence, an alternative antigen delivery approach is required which will induce an effective local immune response in the reproductive tract. Here we discuss the ways in which this might be achieved.

Many of the mammalian sperm surface proteins that have been characterized in recent years have been implicated in sperm--egg interactions and subsequent fertilization. In particular, several members of the MDC protein family, most notably fertilin alpha, fertilin beta and cyritestin (also known as tMDC I), play an important role in sperm--egg interactions in rodents. In light of current data, it is proposed that many of these sperm proteins act co-operatively, and that the concerted effects of several proteins leads to 'maximum fertilizing ability', but that individual proteins are functionally redundant and are therefore not essential. Nevertheless, the co-operative nature of these interactions means that loss of individual proteins might lead to a concomitant reduction in fertilizing ability, a proposal that is supported by our recent finding of non-functional fertilin alpha and tMDC I genes in humans.

The metalloproteinase-like, disintegrin-like, cysteine-rich (MDC) family is a large group of sequence-related proteins, first characterized in the male reproductive tract, but subsequently also identified in non-reproductive tissues. Their primary translation products are of approximately 90 kDa and each can be divided into distinct domains which show remarkable homology to reprolysins; snake venom haemorrhagic components possessing metalloproteinase and/or disintegrin domains. Several MDC proteins are abundantly-expressed in the male reproductive tract, suggesting functions in fertility. We now describe the cloning, sequence determination and characterization of transcripts encoding the human and macaque (Macaca fascicularis) orthologues of a novel member of the MDC family (eMDC II) which is abundantly-expressed in the epididymis. Unlike many MDC proteins expressed in the reproductive tract, eMDC II possesses the extended 'catalytic centre' consensus sequence characteristic of a reprolysin-like metalloproteinase. This suggests that eMDC II has proteolytic activity.

Conference Abstract| February 01 1999 Sequence analysis and characterisation of the rat orthologue of TNFα converting enzyme (TACE) A. J. Beaumont; A. J. Beaumont 1Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK Search for other works by this author on: This Site PubMed Google Scholar J. A. Jury; J. A. Jury 1Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK Search for other works by this author on: This Site PubMed Google Scholar J. Frayne; J. Frayne 1Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK Search for other works by this author on: This Site PubMed Google Scholar R. Devos; R. Devos 3Hoffmann-La Roche, B-9000 Gent, Belgium Search for other works by this author on: This Site PubMed Google Scholar J. Van der Heyden; J. Van der Heyden 3Hoffmann-La Roche, B-9000 Gent, Belgium Search for other works by this author on: This Site PubMed Google Scholar L. Jeffery; L. Jeffery 2Roche Discovery Welwyn, Broadwater Road, Welwyn Garden City, Herts AL7 3AY, UK Search for other works by this author on: This Site PubMed Google Scholar G. J. Price; G. J. Price 2Roche Discovery Welwyn, Broadwater Road, Welwyn Garden City, Herts AL7 3AY, UK Search for other works by this author on: This Site PubMed Google Scholar L. Hall L. Hall 1Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1999) 27 (1): A55. https://doi.org/10.1042/bst027a055 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation A. J. Beaumont, J. A. Jury, J. Frayne, R. Devos, J. Van der Heyden, L. Jeffery, G. J. Price, L. Hall; Sequence analysis and characterisation of the rat orthologue of TNFα converting enzyme (TACE). Biochem Soc Trans 1 February 1999; 27 (1): A55. doi: https://doi.org/10.1042/bst027a055 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1999 Biochemical Society1999 Article PDF first page preview Close Modal You do not currently have access to this content.

Five members of the MDC (metalloproteinase-like,disintegrin-like cysteine-rich domain) family of proteins (fertilin alpha, fertilin beta, tMDC I, tMDC II and tMDC III) are expressed on the surface of macaque (Macaca fascicularis) sperm, where they have been proposed to play a role in sperm-egg binding via an interaction between their disintegrin-like domain and one or more integrins on the egg plasma membrane. Of these, two (fertilin alpha and tMDC I) have recently been shown to be non-functional in the human. Here we report the existence of multiple isoforms of human tMDC II transcripts in the human, all of which are also non-functional owing to the presence of deletions and in-frame termination codons, when compared with the macaque orthologue, a finding which is further supported by the lack of immunoreactivity on Western blots of human testis and sperm extracts probed with a macaque anti-tMDC II polyclonal antiserum. These results are discussed in the context of our proposed model for multiple proteins implicated in sperm-egg interactions.

Mammalian spermatozoa are particularly susceptible to the deleterious effects of reactive oxygen species and lipid peroxidation, which ultimately lead to impaired fertility. A number of enzymes are present in the male reproductive tract which may play a role in preventing oxidative damage; in particular, the epididymis is the site of synthesis and secretion of large amounts of extracellular superoxide dismutase (eSOD). In order to study the distribution of eSOD in the male reproductive tract, and distinguish it from other related superoxide dismutase isoenzymes (e.g. cytosolic SOD), polyclonal antisera have been raised against a recombinant human eSOD fusion protein, expressed in bacterial cells. This protein was expressed from a synthetic gene fragment, using preferred Escherichia coli codons, designed to overcome the problems associated with the high guanine+cytosine content of the natural human eSOD transcript. Using this antiserum, eSOD can be readily detected in a range of human reproductive tissues as well as in human seminal plasma. However, the presence of similar levels of eSOD in the seminal plasma of vasectomized men (probably of prostatic origin) precludes its use as a simple diagnostic indicator of eSOD activity levels in the epididymis.

The sperm surface fertilin complex was first described in the guinea pig where it was found as a heterodimer of alpha and beta subunits, both of which were proposed to play a role in sperm-oolemma recognition and plasma membrane fusion during fertilisation. Whilst the beta subunit is apparently testis-specific, the finding of low levels of fertilin alpha in nonreproductive tissues has cast some doubt on a unique role in fertilisation. Moreover, the absence of a functional fertilin alpha gene in the human would imply that this gene product is not absolutely essential for fertilisation, although it could play a facilitatory role. We now describe the organisation and sequence of the fertilin alpha genes in a range of primates, including the great apes, and find that the gorilla gene, like that of the human, is non-functional. Mel. Reprod. Dev. 51.92-97, 1998. (C) 1998 Wiley-Liss, Inc.