Edward M. Eddy

Haploinsufficiency of protamine-1 or -2 causes infertility in mice

Protamines are the major DNA-binding proteins in the nucleus of sperm in most vertebrates and package the DNA in a volume less than 5% of a somatic cell nucleus. Many mammals have one protamine, but a few species, including humans and mice, have two. Here we use gene targeting to determine if the second protamine provides redundancy to an essential process, or if both protamines are necessary. We disrupted the coding sequence of one allele of either Prm1 or Prm2 in embryonic stem (ES) cells derived from 129-strain mice, and injected them into blastocysts from C57BL/6-strain mice. Male chimeras produced 129-genotype sperm with disrupted Prm1 or Prm2 alleles, but failed to sire offspring carrying the 129 genome. We also found that a decrease in the amount of either protamine disrupts nuclear formation, processing of protamine-2 and normal sperm function. Our studies show that both protamines are essential and that haploinsufficiency caused by a mutation in one allele of Prm1 or Prm2 prevents genetic transmission of both mutant and wild-type alleles.

Protamine 2 Deficiency Leads to Sperm DNA Damage and Embryo Death in Mice

Abstract Cytokinesis is incomplete in spermatogenic cells, and the descendants of each stem cell form a clonal syncytium. As a result, a heterozygous mutation in a gene expressed postmeiotically affects all of the haploid spermatids within a syncytium. Previously, we have found that disruption of one copy of the gene for either protamine 1 (PRM1) or protamine 2 (PRM2) in the mouse results in a reduction in the amount of the respective protein, abnormal processing of PRM2, and inability of male chimeras to transmit either the mutant or wild-type allele derived from the 129-genotype embryonic stem cells to the next generation. Although it is believed that protamines are essential for compaction of the sperm nucleus and to protect the DNA from damage, this has not been proven experimentally. To test the hypothesis that failure of chimeras to transmit the 129 genotype to offspring was due to alterations in the organization and integrity of sperm DNA, we used the single-cell DNA electrophoresis (comet) assay, ultrastructural analysis, and the intracytoplasmic sperm injection (ICSI) procedure. Comet assay demonstrated a direct correlation between the fraction of sperm with haploinsufficiency of PRM2 and the frequency of sperm with damaged DNA. Ultrastructural analysis revealed reduced compaction of the chromatin. ICSI with PRM2-deficient sperm resulted in activation of most metaphase II-arrested mouse eggs, but few were able to develop to the blastocyst stage. These findings suggest that development fails because of damage to paternal DNA and that PRM2 is crucial for maintaining the integrity of sperm chromatin.

5 Gene Expression during Mammalian Meiosis

The expression of a wide variety of genes is developmentally regulated during mammalian meiosis. Drawing mainly on studies in spermatogenesis, this review shows that some of these genes are transcribed exclusively in germ cells, while others are also transcribed in somatic cells. Some of the genes expressed exclusively in spermatogenic cells are unlike any expressed in somatic cells, while others are isologous to genes expressed in somatic cells and are in the same gene family. Some of the developmentally regulated genes also expressed in somatic cells produce spermatogenic cell-specific transcripts, while others produce transcripts that are apparently the same in somatic and germ cells. Possible answers to why so many genes have atypical patterns of expression during meiosis are that: (1) all cell types express certain genes that define their cell type and lineage, (2) spermatogenesis is a developmental process that progresses according to a genetic program directing the sequential and coordinate expression of specific genes. (3) some genes are expressed that encode proteins required for meiosis. (4) some genes are expressed that encode proteins not required until after meiosis, (5) some genes are expressed to compensate for other genes that become inactivated with X chromosome condensation, and (6) it has been suggested that regulation of gene expression becomes leaky during spermatogenesis due to changes in DNA organization, leading to production of irrelevant transcripts. However, it is largely unknown how extrinsic cues from the endocrine system and surrounding somatic cells interact with intrinsic mechanisms of germ cells to activate signal transduction processes regulating transcription during mammalian meiosis.

Expression of the Gene for Mouse Lactate Dehydrogenase C (Ldhc) Is Required for Male Fertility

Abstract The lactate dehydrogenase (LDH) protein family members characteristically are distributed in tissue- and cell type-specific patterns and serve as the terminal enzyme of glycolysis, catalyzing reversible oxidation reduction between pyruvate and lactate. They are present as tetramers, and one family member, LDHC, is abundant in spermatocytes, spermatids, and sperm, but also is found in modest amounts in oocytes. We disrupted the Ldhc gene to determine whether LDHC is required for spermatogenesis, oogenesis, and/or sperm and egg function. The targeted disruption of Ldhc severely impaired fertility in male Ldhc−/− mice but not in female Ldhc−/− mice. Testis and sperm morphology and sperm production appeared to be normal. However, total LDH enzymatic activity was considerably lower in Ldhc−/− sperm than in wild type sperm, indicating that the LDHC homotetramer (LDH-C4) is responsible for most of the LDH activity in sperm. Although initially motile when isolated, there was a more rapid reduction in the level of ATP and in motility in Ldhc−/− sperm than in wild-type sperm. Moreover, Ldhc−/− sperm did not acquire hyperactivated motility, were unable to penetrate the zona pellucida in vitro, and failed to undergo the phosphorylation events characteristic of capacitation. These studies showed that LDHC plays an essential role in maintenance of the processes of glycolysis and ATP production in the flagellum that are required for male fertility and sperm function.

Morphological analysis of germ cell apoptosis during postnatal testis development in normal and Hsp70-2 knockout mice

The present study examined the occurrence of apoptotic cell death in the testis of wild‐type mice from postnatal days 3 to 26 and in juvenile Hsp70‐2 knockout mice. Adult Hsp70‐2 knockout males are infertile and lack spermatids and spermatozoa (Dix et al. [1996a] Proc. Natl. Acad. Sci. U.S.A. 93:3264–3268). To identify the cell types undergoing apoptosis, we also examined the relationship between the occurrence of apoptotic cell death and the expression pattern of the Hsp70‐2 gene product (heat‐shock protein 70‐2 [HSP70‐2]; marker for spermatocytes and spermatids), germ cell nuclear antigen 1 (GCNA1; marker for spermatogonia and spermatocytes), and vimentin (marker for Sertoli cells). This study shows that during postnatal development of the wild‐type mouse testis (1) the percentage of apoptotic cell death detected by the TdT‐mediated dUTP‐biotin nick end labeling (TUNEL) method is higher in mice from days 8 to 22 than in younger or older mice, (2) the majority of apoptotic cells are spermatogonia and less frequently are spermatocytes, and (3) the degenerative cell death of spermatogonia and primary spermatocytes involves apoptosis with fragmentation of DNA. The analysis of apoptotic cell death in the testes of juvenile Hsp70‐2 knockout mice showed an additional increased level of apoptosis at day 17, during the first wave of spermatogenesis, in pachytene spermatocytes. Dev Dyn 208:125–136, 1997.

A-Kinase Anchoring Protein 4 Binding Proteins in the Fibrous Sheath of the Sperm Flagellum

Abstract The fibrous sheath is a unique cytoskeletal structure located in the principal piece of the sperm flagellum and is constructed of two longitudinal columns connected by closely spaced circumferential ribs. Cyclic AMP-dependent protein kinases are secured within specific cytoplasmic domains by A-kinase anchoring proteins (AKAPs), and the most abundant protein in the fibrous sheath is AKAP4. Several other fibrous sheath proteins have been identified, but how the fibrous sheath assembles is not understood. Yeast two-hybrid assays and deletion mutagenesis were used to identify AKAP4-binding proteins and to map the binding regions on AKAP4 and on the proteins identified. We found that AKAP4 binds AKAP3 and two novel spermatogenic cell-specific proteins, Fibrous Sheath Interacting Proteins 1 and 2 (FSIP1, FSIP2). Transcription of Akap4, Akap3, and Fsip1 begins in early spermatid development, whereas transcription of Fsip2 begins in late spermatocyte development. AKAP3 is synthesized in round spermatids and incorporated into the fibrous sheath concurrently with formation of the rib precursors. However, AKAP4 is synthesized and incorporated into the nascent fibrous sheath late in spermatid development. The AKAP4 precursor is processed in the flagellum and only the mature form of AKAP4 appears to bind AKAP3. These results suggest that AKAP3 is involved in organizing the basic structure of the fibrous sheath, whereas AKAP4 has a major role in completing fibrous sheath assembly.

Spermatogenic Cells Do Not Require Estrogen Receptor-α for Development or Function

Estrogen receptors alpha (ERα) and beta (ERβ) are ligand-dependent transcription factors and members of the nuclear hormone receptor superfamily encoded by separate genes. Male mice homozygous for a mutation in the gene encoding ERα are infertile. To determine whether germ cells or somatic cells require ERα, germ cells were transplanted from donor males homozygous for the mutation (ERα−/−) to testes of wild-type (ERα+/+) recipient mice depleted of germ cells. The recipients served as “surrogate fathers” for the infertile ERα−/− males. When mated to wild-type females, the recipients sired offspring heterozygous for the mutation (ER+/−) and carrying the coat-color marker of the ERα−/− donor mice. These studies show that male germ cells do not require ERα for development or to function in fertilization, and imply that male ERα−/− mice are infertile due to disruption of estrogen action within somatic cells of the male reproductive system.

Phosphoglycerate Kinase 2 (PGK2) Is Essential for Sperm Function and Male Fertility in Mice

Abstract Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2−/− and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs−/− males, the Pgk2−/− males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2−/− mice may provide further insights into the regulation of sperm metabolism.

Impaired sperm fertilizing ability in mice lacking Cysteine-RIch Secretory Protein 1 (CRISP1)

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. Epididymal protein CRISP1, a member of the Cysteine-RIch Secretory Protein (CRISP) family, was identified by our laboratory and postulated to participate in both sperm-zona pellucida (ZP) interaction and gamete fusion by binding to egg-complementary sites. To elucidate the functional role of CRISP1 in vivo, we disrupted the Crisp1 gene and evaluated the effect on animal fertility and several sperm parameters. Male and female Crisp1(-/-) animals exhibited no differences in fertility compared to controls. Sperm motility and the ability to undergo a spontaneous or progesterone-induced acrosome reaction were neither affected in Crisp1(-/-) mice. However, the level of protein tyrosine phosphorylation during capacitation was clearly lower in mutant sperm than in controls. In vitro fertilization assays showed that Crisp1(-/-) sperm also exhibited a significantly reduced ability to penetrate both ZP-intact and ZP-free eggs. Moreover, when ZP-free eggs were simultaneously inseminated with Crisp1(+/+) and Crisp1(-/-) sperm in a competition assay, the mutant sperm exhibited a greater disadvantage in their fusion ability. Finally, the finding that the fusion ability of Crisp1(-/-) sperm was further inhibited by the presence of CRISP1 or CRISP2 during gamete co-incubation, supports that another CRISP cooperates with CRISP1 during fertilization and might compensate for its lack in the mutant mice. Together, these results indicate that CRISP proteins are players in the mammalian fertilization process. To our knowledge this is the first knockout mice generated for a CRISP protein. The information obtained might have important functional implications for other members of the widely distributed and evolutionarily conserved CRISP family.

Mouse spermatogenic cell–specific type 1 hexokinase (mHk1-s) transcripts are expressed by alternative splicing from the mHk1 gene and the HK1-S protein is localized mainly in the sperm tail

Unique type 1 hexokinase (HK1) mRNAs are present in mouse spermatogenic cells (mHk1‐s). They encode a spermatogenic cell–specific sequence region (SSR) but not the porin‐binding domain (PBD) necessary for HK1 binding to porin on the outer mitochondrial membrane. This study determined the origin of the multiple Hk1‐s transcripts in mouse spermatogenic cells and verified that they are translated in mouse spermatogenic cells. It also showed that a single mHk1 gene encodes the mHk1 transcripts of somatic cells and the mHk1‐sa and mHk1‐sb transcripts of spermatogenic cells, that alternative exons are used during mHk1 gene expression in mouse spermatogenic cells, and that mHK1‐S is translated in mouse spermatogenic cells and is localized mainly with the fibrous sheath in the tail region, not with the mitochondria in the midpiece of mouse sperm. Mol. Reprod. Dev. 49:374–385, 1998.