Meiosis I arrest abnormalities lead to severe oligozoospermia in meiosis 1 arresting protein (M1ap)-deficient mice

Abstract

Meiosis 1 arresting protein (M1ap) is a novel vertebrate gene expressed exclusively in germ cells of the embryonic ovary and the adult testis. In male mice, M1ap expression, which is present from spermatogonia to secondary spermatocytes, is evolutionarily conserved and has a specific spatial and temporal pattern suggestive of a role during germ cell development. To test its function, mice deficient in M1ap were created. Whereas females had histologically normal ovaries, males exhibited reduced testicular size and a myriad of tubular defects, which led to severe oligozoospermia and infertility. Although some germ cells arrested at the zygotene/pachytene stages, most cells advanced to metaphase I before arresting and entering apoptosis. Cells that reached metaphase I were unable to properly align their chromosomes at the metaphase plate due to abnormal chromosome synapses and failure to form crossover foci. Depending on the state of tubular degeneration, all germ cells, with the exemption of spermatogonia, disappeared; with further deterioration, tubules displaying only Sertoli cells reminiscent of Sertoli cell-only syndrome in humans were observed. Our results uncovered an essential role for M1ap as a novel germ cell gene not previously implicated in male germ cell development and suggest that mutations in M1AP could account for some cases of nonobstructive oligozoospermia in men.

FIG. 1

M1ap expression and gene disruption strategy. A) RT-PCR analyses of postnatal testes showing the onset of M1ap expression by P7. Gapdh was used as loading control. B) Quantitative RT-PCR analyses of M1ap expression in specific spermatogenic cell types. The highest expression of M1ap is seen in P8 type B spermatogonia and spermatids of adult mice. A, type A spermatogonia; AES/RB, adult elongated spermatids/residual bodies; AP, adult pachytene; ARS, adult round spermatids; B, type B spermatogonia; JP, juvenile pachytene; L/Z, leptotene/zygotene; PA, primitive type A spermatogonia; PL, preleptotene; S, Sertoli cells. C) Gene disruption by gene-trapping methods. The mutant allele (M1ap^lz) was produced by inserting a β-geo cassette into intron 6. The β-geo cassette contains a splice acceptor (SA) capable of fusing to the splice donor of the preceding exon. A, B, and C represent the position within the genome of the primers used for mouse genotyping. ATG, start codon; TAA, stop codon. D) Genotyping strategy. Primers A, B, and C were used in a single PCR reaction. Wild-type allele = 358 bp; mutant allele = 296 bp. E) Western blot showing different levels of M1AP protein expression (∼53 kDa) in wild-type (+/+) and M1ap^lz/lz testes. β-Actin was used as loading control.

FIG. 2

Decreased testicular weight and multiple tubular defects in M1ap-deficient mice. A) Testes from M1ap^lz/lz mice exhibit an approximately 50% reduction in weight compared to wild-type and heterozygous mice. B–I) Multiple histological abnormalities in adult M1ap^lz/lz testes. B and C) Sections of wild-type (+/+) testis and corresponding epididymis showing normal spermatogenesis. Inset in B shows metaphase cell. D–F) M1ap^lz/lz testis sections illustrating abnormal metaphase I cells (arrows), vacuolization (asterisk), and an overall reduction in secondary spermatocytes and tubular diameter. The drastic decrease in secondary spermatocytes is evident in the corresponding epididymis, where very few elongated spermatids are found. Round spermatocytes are also visible, some of which show characteristics of apoptosis (arrowheads in F). Insets in D and E show misaligned chromosomes at metaphase I plate. G and H) M1ap^lz/lz testis sections detailing tubules containing no germ cells beyond zygotene stages (white arrows), vacuolization (asterisk), Sertoli cell clusters in the center of the tubule (white arrowhead), and SCO tubules (&). I) No spermatozoa are detected in the corresponding epididymis. Bar = 100 μm.

FIG. 3

Chronological analyses of M1ap^lz/lz mice revealed testicular defects as early as P14. A) Hematoxylin and eosin-Y-stained sections of P14 M1ap^lz/lz testes illustrating tubules devoid of meiotic germ cells (asterisks). IF showing a decrease of MVH-positive (green) and GCNA1-positive (red) cells (&). B) Sections of P21 M1ap^lz/lz testes showing tubules with no meiotic germ cells beyond zygotene/pachytene stages (black asterisks), metaphase I arrested cells (arrows), and tubules containing only Sertoli cells and spermatogonia (&). A dramatic decrease is seen in cells expressing MVH, along with an abnormal distribution of GCNA1-positive cells (white asterisks). C) Sections of P28 M1ap^lz/lz testes depicting multiple tubular defects. Some tubules are devoid of germ cells beyond the zygotene/pachytene stages (asterisk), whereas others have an abundance of abnormal metaphase I cells (arrows), all of which lead to a lack of secondary spermatocytes (SS). MVH and GCNA1 staining revealed a disorganized germinal epithelium containing degenerating tubules (&), reduced tubular diameter, and an absence of secondary spermatocytes (SS). D) Sections of P56 M1ap^lz/lz testes showing a dramatic reduction in secondary spermatocytes (SS), abnormal metaphase I cells (arrows), SCO tubules (asterisks), and reduced tubular diameter. In addition, a decrease in germ cells expressing MVH and abnormal localization of GCNA1-positive germ cells (arrowheads) is seen. Bar = 100 μm.

FIG. 4

Localization of γH2AX and SYCP3 protein in wild-type (+/+) and M1ap^lz/lz spermatocytes. In wild-type spermatocytes, γH2AX disappears from autosomes by pachynema, whereas it remains in association with the XY body until the end of diplonema. In M1ap^lz/lz spermatocytes, γH2AX signal is retained in the autosomes as late as diplonema. Nonhomologous pairing (arrowhead) and separate X-Y chromosomes are also commonly observed. Bar = 100 μm.

FIG. 5

Failed chromosome synapses in M1ap^lz/lz spermatocytes. Whereas all chromosomes from wild-type (+/+) cells show SYCP1 signal (red) indicative of proper homologous pairing during pachynema, a proportion of chromosomes in M1ap^lz/lz cells failed to synapse (arrows). Bar = 100 μm.

FIG. 6

Failed formation of crossover foci and increased apoptosis in M1ap^lz/lz germ cells. A) M1ap^lz/lz pachytene spermatocytes are deficient in MLH1 foci assembly (red). Original magnification ×1000. B) TUNEL assays revealed increased apoptosis in M1ap^lz/lz testes. Bar = 100 μm.