Paula E. Cohen

MEIOTIC PACHYTENE ARREST IN MLH1-DEFICIENT MICE

Germ line mutations in DNA mismatch repair genes including MLH1 cause hereditary nonpolyposis colon cancer. To understand the role of MLH1 in normal growth and development, we generated mice that have a null mutation of this gene. Mice homozygous for this mutation show a replication error phenotype, and extracts of these cells are deficient in mismatch repair activity. Homozygous mutant males show normal mating behavior but have no detectable mature sperm. Examination of meiosis in these males reveals that the cells enter meiotic prophase and arrest at pachytene. Homozygous mutant females have normal estrous cycles and reproductive and mating behavior but are infertile. The phenotypes of the mlh1 mutant mice are distinct from those deficient in msh2 and pms2. The different phenotypes of the three types of mutant mice suggest that these three genes may have independent functions in mammalian meiosis.

Mutation in the mismatch repair gene Msh6 causes cancer susceptibility

Mice carrying a null mutation in the mismatch repair gene Msh6 were generated by gene targeting. Cells that were homozygous for the mutation did not produce any detectable MSH6 protein, and extracts prepared from these cells were defective for repair of single nucleotide mismatches. Repair of 1, 2, and 4 nucleotide insertion/deletion mismatches was unaffected. Mice that were homozygous for the mutation had a reduced life span. The mice developed a spectrum of tumors, the most predominant of which were gastrointestinal tumors and B- as well as T-cell lymphomas. The tumors did not show any microsatellite instability. We conclude that MSH6 mutations, like those in some other members of the family of mismatch repair genes, lead to cancer susceptibility, and germline mutations in this gene may be associated with a cancer predisposition syndrome that does not show microsatellite instability.

Mammalian MutS homologue 5 is required for chromosome pairing in meiosis

MSH5 (MutS homologue 5) is a member of a family of proteins known to be involved in DNA mismatch repair. Germline mutations in MSH2, MLH1 and GTBP (also known as MSH6) cause hereditary non–polyposis colon cancer (HNPCC) or Lynch syndrome. Inactivation of Msh2, Mlh1, Gtmbp (also known as Msh6) or Pms2 in mice leads to hereditary predisposition to intestinal and other cancers. Early studies in yeast revealed a role for some of these proteins, including Msh5, in meiosis. Gene targeting studies in mice confirmed roles for Mlh1 and Pms2 in mammalian meiosis. To assess the role of Msh5 in mammals, we generated and characterized mice with a null mutation in Msh5. Msh5–/– mice are viable but sterile. Meiosis in these mice is affected due to the disruption of chromosome pairing in prophase I. We found that this meiotic failure leads to a diminution in testicular size and a complete loss of ovarian structures. Our results show that normal Msh5 function is essential for meiotic progression and, in females, gonadal maintenance.

Meiotic arrest and aneuploidy in MLH3-deficient mice.

MutL homolog 3 (Mlh3) is a member of a family of proteins conserved during evolution and having dual roles in DNA mismatch repair and meiosis. The pathway in eukaryotes consists of the DNA-binding components, which are the homologs of the bacterial MutS protein (MSH 2–6), and the MutL homologs, which bind to the MutS homologs and are essential for the repair process. Three of the six homologs of MutS that function in these processes, Msh2, Msh3 and Msh6, are involved in the mismatch repair of mutations, frameshifts and replication errors, and two others, Msh4 and Msh5, have specific roles in meiosis. Of the four MutL homologs, Mlh1, Mlh3, Pms1 and Pms2, three are involved in mismatch repair and at least two, Pms2 and Mlh1, are essential for meiotic progression in both yeast and mice. To assess the role of Mlh3 in mammalian meiosis, we have generated and characterized Mlh3−/− mice. Here we show that Mlh3−/− mice are viable but sterile. Mlh3 is required for Mlh1 binding to meiotic chromosomes and localizes to meiotic chromosomes from the mid pachynema stage of prophase I. Mlh3−/− spermatocytes reach metaphase before succumbing to apoptosis, but oocytes fail to complete meiosis I after fertilization. Our results show that Mlh3 has an essential and distinct role in mammalian meiosis.

MACROPHAGES: IMPORTANT ACCESSORY CELLS FOR REPRODUCTIVE FUNCTION

Macrophages are found throughout reproductive tissues. To determine their role(s), we have studied mice homozygous for a null mutation (Csfmop) in the gene encoding the major macrophage growth factor, colony‐stimulating factor‐1 (CSF‐1). Both male and female Csfmop/Csfmop mice have fertility defects. Males have low sperm number and libido as a consequence of dramatically reduced circulating testosterone. Females have extended estrous cycles and poor ovulation rates. CSF‐1 is the principal growth factor regulating macrophage populations in the testis, male accessory glands, ovary, and uterus. However, analyses of CSF‐1 nullizygous mice suggest that the primary reproductive defect is in the development of feedback regulation of the hypothalamic‐pituitary axis. Although not correlating with deficiencies of microglia populations, electrophysiological investigations indicate an impairment of neuronal responses. This suggests that microglia, under the influence of CSF‐1, act to organize neuronal connectivity during development and that the absence of this function results in a perturbation of the hypothalamicpituitary‐gonadal axis. Macrophages also appear to have functions in the differentiated tissues of the reproductive system, including having a positive influence on steroidogenic cells. These data suggest that macrophages, through their trophic functions, can be considered as essential accessory cells for normal reproductive functioning. J. Leukoc. Biol. 66:765–772; 1999.

Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18(Ink4c) and p19(Ink4d).

ABSTRACT Male mice lacking both the Ink4c and Ink4dgenes, which encode two inhibitors of D-type cyclin-dependent kinases (Cdks), are infertile, whereas female fecundity is unaffected. Both p18Ink4c and p19Ink4d are expressed in the seminiferous tubules of postnatal wild-type mice, being largely confined to postmitotic spermatocytes undergoing meiosis. Their combined loss is associated with the delayed exit of spermatogonia from the mitotic cell cycle, leading to the retarded appearance of meiotic cells that do not properly differentiate and instead undergo apoptosis at an increased frequency. As a result, mice lacking bothInk4c and Ink4d produce few mature sperm, and the residual spermatozoa have reduced motility and decreased viability. Whether or not Ink4d is present, animals lackingInk4c develop hyperplasia of interstitial testicular Leydig cells, which produce reduced levels of testosterone. The anterior pituitary of fertile mice lacking Ink4c or infertile mice doubly deficient for Ink4c and Ink4d produces normal levels of luteinizing hormone (LH). Therefore, the failure of Leydig cells to produce testosterone is not secondary to defects in LH production, and reduced testosterone levels do not account for infertility in the doubly deficient strain. By contrast,Ink4d-null or double-null mice produce elevated levels of follicle-stimulating hormone (FSH). Because Ink4d-null mice are fertile, increased FSH production by the anterior pituitary is also unlikely to contribute to the sterility observed inInk4c/Ink4d double-null males. Our data indicate that p18Ink4c and p19Ink4d are essential for male fertility. These two Cdk inhibitors collaborate in regulating spermatogenesis, helping to ensure mitotic exit and the normal meiotic maturation of spermatocytes.

Use of the Osteopetrotic Mouse for Studying Macrophages in the Reproductive Tract

Macrophages are found at all levels of the reproductive tract in both males and females. They represent a constitutive cellular component of some tissues, such as the interstitium of the testis, while in other tissues their numbers may fluctuate at specific times, as is the case for the uterus. The increase in macrophage numbers in the uterus and other tissues—and their subsequent activation—is known to be regulated by the steroid hormones, estrogen and progesterone (1), and by polypeptide growth factors. These growth factors include colony stimulating factor I (CSF-I), also known as macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), transforming growth factor β1 (TGFβ1), tumor necrosis factor a (TNFα), and interferon γ (IFNγ). Moreover, there is significant evidence, in the uterus at least, to suggest that production of these growth factors in target tissues is steroid hormone regulated. Thus, it appears that steroid-induced events in the reproductive tract may be mediated by macrophages via their steroid-induced chemoattractants and growth factors.