The small GTPase Rheb is required for spermatogenesis but not oogenesis

Abstract

The process of germ cell development is under the tight control of various signaling pathways, among which the PI3K-Akt-mTOR pathway is of critical importance. Previous studies have demonstrated sex-specific roles for several components of this pathway. In the current study, we aimed to evaluate the role of Rheb, a member of the small GTPase superfamily and a critical component for mTORC1 activation, in male and female gametogenesis. The function of Rheb in development and the nervous system has been extensively studied, but little is known about its role in the germ line. We have exploited genetic approaches in the mouse to study the role of Rheb in the germ line and have identified an essential role in spermatogenesis. Conditional knockout (cKO) of Rheb in the male germ line resulted in severe oligoasthenoteratozoospermia and male sterility. More detailed phenotypic analyses uncovered an age-dependent meiotic progression defect combined with subsequent abnormalities in spermiogenesis as evidenced by abnormal sperm morphology. In the female, however, germ-cell specific inactivation of Rheb was not associated with any discernible abnormality; these cKO mice were fertile with morphologically unremarkable ovaries, normal primordial follicle formation, and subsequent follicle maturation. The absence of an abnormal ovarian phenotype is striking given previous studies demonstrating a critical role for the mTORC1 pathway in the maintenance of primordial follicle pool. In conclusion, our findings demonstrate an essential role of Rheb in diverse aspects of spermatogenesis but suggest the existence of functionally redundant factors that can compensate for Rheb deficiency within oocytes.

Figure 1. RNA expression analysis of Rheb and RhebL1

Graphs show relative expression levels of single probe set for indicated gene across multiple samples; error bars represent SEM. Samples are (left to right) Foxo3 +/+ PD1, -3, -7, -14 (black), Foxo3 −/− PD1, -3, -7, -14 (red), adult ovary, laser-capture microdissected (LCM) primary and secondary oocytes, LCM somatic cells (primary plus secondary granulosa cells and surrounding stroma), eggs, cumulus cells, ES cells, normal adult testis, Sl/Sld (germ cell-depleted) adult testis, Foxo3 +/+ E11 embryos, Foxo3 −/− E11 embryos, adrenal gland, placenta, uterus, bone marrow, spleen, thymus, brain, eye, skeletal muscle, heart, intestine, kidney, liver, and lung. (A) Vasa/Ddx4 as representative control (germ cell-specific gene in both males and females). (B) Rheb. (C) RhebL1 (probe set #1). (D) RhebL1 (probe set #2).

Figure 2. Genotype confirmation of germ cell Rheb conditional knockout

(A) An ethidium bromide-stained 1% agarose gel shows the presence of the Rheb-floxed allele (850 bp) in tail DNA from a Rheb homozygous floxed mouse as well as all other tissues of a Vasa-Cre; Rheb^f/+ (heterozygous) male. The floxed (f) band is decreased in the Vasa-Cre; Rheb^f/+ testis consistent with Vasa-Cre mediated recombination in germ cells. The wt (+) band (650bp) is observed in tissues from this heterozygous male mouse, as expected. (B) Cre-mediated excision was further confirmed by the presence of the Rheb null allele (650bp) in a Vasa-Cre; Rheb^f/+ testis as well as a Rheb^f/− control testis. The Rheb null allele was not detected in other tissues (e.g. skin, lung, and spleen). NS=non-specific PCR product (~750bp).

Figure 3. Conditional Rheb inactivation results in a progressive defect of spermatogenesis

(A) Intact testes. Right=Vasa-Cre; Rheb^f/−; Left=Rheb^f/− sibling control. Ruler markings = 1 mm. (B) Testis weights in mice of different ages. Red=Vasa-Cre; Rheb^f/−; Blue=Rheb^f/− sibling control. *P < 0.05, **P < 0.005, by unpaired T test (two months, n = 2; three months, n = 3; four months, n = 1 [no statistical test performed]; five months, n = 3) (C) H&E stained tissue sections from testes of two to five month-old Rheb cKO and sibling control mice. Representative fields illustrate an abnormal organization of seminiferous tubules at the two month time point and the age dependent defect in meiotic progression resulting in the progressive loss of round and elongating spermatids in the 5 month old testis. Arrowheads indicate large multinucleated germ cells. Bar=50 μm.

Figure 4. Ovaries of Rheb cKO mice are grossly normal and contain growing follicles of all stages as well as morphologically-normal primordial follicles

(A) Intact ovaries of Rheb cKO (Vasa-Cre; Rheb^f/−) females were of similar size and shape relative to sibling (Rheb^f/−) controls. Bar = 1 mm. (B) H&E stained tissue sections from Rheb cKO and sibling control ovaries revealed growing follicles of all stages. Bar = 250 μm. (C) Higher magnification shows growing primary and secondary follicles. Insets: morphologically normal, quiescent, primordial follicles. Scale bar = 50 μm.

Figure 5. Total sperm production is drastically decreased and remaining sperm are malformed

(A) Histology of epididymides from control and experimental mice. Bar = 100 μm. (B) The decrease in epididymal sperm was quantified by sperm head density counts (n = 1 for all time points and genotypes). (C) Total sperm counts were performed after releasing the sperm from the epididymis. ***P < 0.0001. Red=Vasa-Cre; Rheb^f/− n = 4; Blue=Rheb^f/− n = 5 sibling controls. (D) The few sperm obtained from the epididymis of Rheb cKO mice were all immotile and exhibited abnormal morphology. Bar = 10 μm; all panels at same magnification.

Figure 6. Serial breeding assays reveal that Rheb cKO males are sterile while Rheb cKO females are fertile with normal fecundity to 18 weeks of age

(A) Aggregate (i.e. all animals of the same genotype averaged) and (B) Individual (i.e. every line represents a single animal) representations of total progeny per female. (B) Aggregate and (C) Individual representations of total progeny per male.

Figure 7. Cell counts reveal meiotic progression defect

(A) Foxo1 and (B) Crem1 immunostains of Rheb cKO (Vasa-Cre; Rheb^f/−) and control testes at two and five months of age. Bar = 50 μm and 100 μm, respectively. (C) Quantification of Foxo1 and Crem1 positive cell counts in twenty tubules; *P < 0.05, **P < 0.0005 by unpaired T test. (D) Counts of spermatocytes and spermatids in hematoxylin-stained tissue sections. The counts of elongated spermatids closely mirrored the observed decrease in Crem1⁺ round spermatids; *P < 0.05, **P < 0.005, ***P < 0.0005, by unpaired T test.

Figure 8. Analyses of cell proliferation and meiotic entry

(A–H) Immunostaining for Ki67 as a marker for cellular proliferation. Bar=100 μm (I–P) Immunostaining for pH2AX as a marker of meiotic entry. Scattered apoptotic germ cells server as an internal positive control (arrowheads) since apoptotic cells undergo DNA fragmentation and are strongly positive for pH2AX. Bar = 25 μm.

Figure 9. Analysis of mTOR and downstream effectors

Immunostains with phosphorylation-site specific antibodies against canonical mTOR pathway members in both ovaries (left) and testes (right). Bar = 250 μm and 100 μm, respectively. (A–D) Total mTOR. (E–H) p-mTOR. (I–L) p-S6K. (M–P) p-4EBP.