Following embryonic sex determination in mice (~E10.5), male and female germ cells establish genetic hierarchies to maintain germ cell identity during mitotic proliferation, meiosis, and maturation. By comparing protein profiles of normal versus Figla
null ovaries and by ectopically expressing FIGLA in male germ cells, we have provided evidence that Figla
, initially implicated as an activator of oocyte-specific genetic hierarchies (28
), also inhibits sperm-associated developmental pathways during postnatal oogenesis.
Ectopic expression of Figla
in male germ cells in transgenic mice downregulates a set of testis-associated genes that were initially identified by upregulation in Figla
null newborn ovaries. The consequent testicular pathology and pre- and postmeiotic effects on spermatogenesis result in male sterility and suggest a role for FIGLA in the downregulation of male-associated genes during oogenesis, when Figla
is normally expressed. Nanos2
also has a sexually dimorphic effect on gene expression, although its effect is earlier in gametogenesis and in the opposite sex. Nanos2
encodes an RNA binding protein first observed in germ cells within the colonized gonad and, in its absence, most male germ cells undergo apoptosis (50
). Expression levels of both Figla
, an oocyte-specific homeobox transcription factor (36
), were increased in Nanos
null male gonads at E16.5. To confirm the effect, Nanos2
was ectopically expressed in female cells, where it suppressed Stra8
expression and inhibited meiosis but also activated several male genes, including Tdrd1
(DNA cytosine-5-methyltransferase 3-like) during embryogenesis (47
is required for maintenance of stem cells as premeiotic spermatogonia (42
), whereas Figla
regulates events in growing oocytes that are arrested in the prophase of the first meiotic division (46
), well after the establishment of sexual identity. Nevertheless, both Figla
appear to support female and male germ cell developmental programs, respectively, at least in part by suppressing genetic hierarchies promoting the developmental programs of the opposite sex.
The observed absence of male germ cell gene expression within normal oocytes could result from the lack of activation or from repression of testis genes. While limiting access to DNA binding sites (e.g., nucleosome positioning, histone modifications, DNA methylation) can prevent gene activation, it would not account for the ability of ectopically expressed FIGLA to repress male genes during spermatogenesis. However, changes in dimerization partners or independent cofactors that interact with FIGLA could prevent expression of testis genes during oogenesis. These effects could be direct or indirect. Myogenic regulatory factors (MRFs), a family of canonical bHLH transcription factors, are instructive and can both activate and repress downstream target genes (49
), depending on dimerization partners (30
), epigenetic modifications (14
), and recruitment of additional proteins to the transcriptional complex (4
). FIGLA is likely to utilize a similar strategic repertoire for its physiological effects during oogenesis.
The developmental imperative of spermatogenesis is the production of haploid gametes capable of fertilization, which is necessary for propagation of the species. Following onset of sexual maturity, TSPY1-Figla transgenic male mice were initially fertile, but the intervals between litters became delayed, and none of the male mice sired litters after 5 months of mating. Figla was robustly expressed in male germ cells by 3 weeks of age in TSPY1-Figla transgenic mice, and decreased testicular size by 2 months was accompanied by repression of at least seven testis-associated genes. However, significant abnormalities in spermatogenesis were not observed until 5 months. The initial delay and progressive nature in the severity of the phenotype could have several etiologies. Figla encodes a transcription factor, and therefore its presence is manifest only secondarily through the repression of target genes. At least some of these target genes also encode transcription factors which, while affecting a broader network of genes, could further delay appearance of the full phenotype. In addition, Figla is not fully expressed until several weeks after birth, and depending on the stability of cognate mRNA and protein the phenotype might not become evident until gene products are degraded below a functional threshold.
Multiple molecular defects of spermatogenesis were observed in the transgenic mice (Table ). Tnp2
, which was upregulated in Figla
null ovaries, was less abundant, as were Prm1
, in TSPY1-Figla
transgenic male mice. During normal spermatogenesis, DNA is repackaged from somatic histones via transition proteins (TNP1 and TNP2) onto protamines (PRM1 and PRM2) to form a highly condensed nucleus characteristic of the mature male gamete. Males lacking TNP2 have decreased fecundity (depending on genetic background) and defects in spermatogenesis, including incomplete chromatin condensation, sperm head abnormalities, and reduced sperm motility (1
). Mice that are haploid insufficient in either protamine 1 or 2 have normal sperm counts, but dysmorphology and altered chromatin integrity results in a sterile phenotype (8
). A similar sterile phenotype with haploid insufficiency is observed in chimeric mice established from embryonic stem cells with a null allele of Cyp17a1
(cytochrome P450, family 17, subfamily a, polypeptide 1) (29
), which is also upregulated in Figla
null ovaries. All four of these genes affect the later stages of spermatogenesis with defects in morphology and motility, and sperm with the Cyp17a1
null allele have the same severe tail angulation observed with TSPY1-Figla
The infertility observed in TSPY1-Figla transgenic male mice arises both from defective spermatogenesis and decreased sperm production in older mice. Spermatogenic cell apoptosis was detected by a TUNEL assay beginning in 4-month-old mouse testes and mostly affected spermatogonia germ cells. However, cell death of stem cells may not fully account for decreased sperm counts in older TSPY1-Figla mice, which also have widespread and progressive testicular degeneration beginning at 5 months. Few elongated spermatids were detected in stage XI and XII seminiferous tubules and there was a dramatic decrease in epididymal sperm, which had concomitant defects in sperm motility and apparent abnormalities in sperm volume regulation. The cumulative effect of these abnormalities in spermatogenesis affects normal gamete development and causes a striking reduction in rates of successful fertilization in vitro and in vivo.
Perhaps most striking, ectopic expression of Figla
severely disrupts the chromatoid body, a cytoplasmic structure characteristic of postmeiotic spermatids. It is derived from the nuage, which are granulated, acellular, cytoplasmic structures interspersed among mitochondria in both growing oocytes and in the early stages of spermatogenesis. In male germ cells, the chromatoid body is defined as an electron-dense structure initially scattered in the cytoplasm of spermatocytes which becomes condensed as a single structure in round spermatids and persists in elongating haploid spermatids prior to sequestration in the residual body (12
). Specific RNA and proteins have been associated with this structure, including MVH, MILI, TDRD1, TDRD6, and TDRD7 (18
). Proteins encoded by Mvh
interact with one another, and disruption of either gene blocks spermatogenesis in the zygotene or early pachytene stage of MI prophase (24
). TDRD1, TDRD6, and TDRD7 also interact with one another, and their localization to the chromatoid body requires the presence of MVH (18
). More recent data have demonstrated that TDRD1 interacts with MILI, and the absence of TDRD1 in null mice activates germ line transposons and affects the profile of piRNAs (41
). TDRD6 also binds to MILI and MIWI (official name, Piwi1
), and altered miRNA expression profiles are observed in Tdrd6
null testes (52
). In addition, both TDRD1 and TDRD6 interact with MVH, and mice lacking either protein have small testes with disorganized spermatogenesis and male sterility. Tdrd1
null testes have meiotic defects in prophase I, whereas Tdrd6
null testes are devoid of elongated spermatids. Although the chromatoid body is present in normal round spermatids, it is significantly disrupted in both Tdrd1
null male mice (10
FIGLA, ectopically expressed in male germ cells, effectively downregulates Tdrd1, Tdrd6, and Tdrd7, leading to a phenotype that encompasses those described in Tdrd1 and Tdrd6 null mice, including male sterility, meiotic defects in diakinesis/metaphase stages, a postmeiotic defect in elongated round spermatids, loss of the chromatoid body structure, and displacement of MVH and MILI proteins. The absence of the chromatoid body integrity and mislocalization of the chromatoid body-associated proteins in TSPY1-Figla transgenic testes provide further evidence that TDRD proteins are required for spermatogenesis and chromatoid body architecture. Thus, our study provides evidence that FIGLA represses multiple male germ cell-associated genes, the expression of which could disrupt normal oogenesis.
These data are consistent with FIGLA, a bHLH transcription factor, sustaining the female phenotype by activating female and repressing male germ cell genetic hierarchies in growing oocytes during postnatal ovarian development. Combining the gain of function of TSPY1-Figla transgenic males with the loss of function of Figla null females provides complementary models for further analysis of the genetic hierarchies that maintain a female-specific phenotype during oogenesis.