Datos y Definición de las Variables

To study the function of autophagy in Sertoli cells I generated mice with conditional knockout (cKO) alleles for Becn1 in adult Sertoli cells. Becn1^fl/fl mice were crossed with anti-Müllerian hormone receptor 2-Cre (Amhr2tm3(cre)Bhr) mice to generate male and female mice of genotype Amhr2tm(cre)Bhr/+; Becn1^fl/fl or Becn1 cKOs (Gawriluk et al. 2011;

Jamin et al. 2002). The Amhr2-Cre allele is a knock-in to the Amhr2 locus and thus, is expressed in cells that normally express Amhr2. This includes the Müllerian duct mesenchyme, embryonic Leydig and adult Sertoli cells in the male (Jamin et al. 2002;

Tanwar et al. 2010; Jeyasuria et al. 2004). Wildtype (WT) mice were siblings of Becn1 cKO mice that did not carry Cre. Becn1 cKO mice were born at regular Mendelian frequencies and were healthy, with the exception of infertility in males and subfertility in females. The Becn1 cKO females had smaller litters and hypertrophy in the uterus and oviducts (data not shown). For the remainder of this chapter I will refer only to the males. Becn1 cKO males were mated to CAG-CAT-EGFP Cre-reporter mice to test the Sertoli cell deletion specificity (Kawamoto et al. 2000). In cells that have had Cre-recombinase expressed, the CAT cassette is removed and an EGFP cassette is

expressed. EGFP was detected by epi-fluorescence in the seminiferous tubules of testis from CAG-CAT-EGFP⁺; Becn1 cKO mice but not testis from CAG-CAT-EGFP⁺; WT mice (Figure 4.1 A, B). Immunofluorescence for EGFP on fixed tissue sections indicates EGFP expression only in what appears to be the Sertoli cells of 6-week old Becn1 cKO testis (Figure 4.1 C, D). Previous studies have used the Amhr2-Cre mouse to study the Leydig cell population (Jeyasuria et al. 2004; Tanwar et al. 2010); however, I did not detect EGFP expression in the interstitial cells, suggesting that recombination does not occur in the Leydig cells. Thus, Amhr2-Cre expression is specific to the adult Sertoli cells in six-week-old testes.

148

To determine extent of knockout I performed immunoblotting to quantify the protein amount of BECN1 and SQSTM1 relative to ACTIN on whole testis from 6- and 10-week-old WT and Becn1 cKO mice. There was no difference in BECN1 quantity between 6-week-old WT and Becn1 cKO (p=0.6, Holm-Sidak post hoc test) (Figure 4.7 A, B).

However, BECN1 was reduced by 2-fold in Becn1 cKO compared to WT at 10-weeks-old (p<0.01, Holm-Sidak post hoc test), demonstrating knockout (Figure 4.7 A, B). The 10-week-old Becn1 cKO group probably is a better reflection of deletion rate than the 6-week-old Becn1 cKO, which has a greater percentage of germ cells in the seminiferous tubules. The protein-turnover of SQSTM1, also known as p62, is autophagy-dependent and can be used to monitor autophagy, such that an accumulation of SQSTM1 indicates inhibition of autophagy and vice-versa (Bjørkøy et al. 2005). There was no difference in SQSTM1 at 6-weeks between WT and Becn1 cKO (p=0.8, Holm-Sidak post hoc test) (Figure 4.7 A, B). However, at 10-weeks, SQSTM1 was increased by 2-fold in Becn1 cKO compared to WT (p<0.01, Holm-Sidak post hoc test), demonstrating the

accumulation of SQSTM1. Therefore, BECN1 is reduced and there is inhibition of autophagy due to the increase in SQSTM1 in Becn1 cKO testes.

To determine the effects of Becn1 deletion on fertility I set up a fertility assay, as described in the materials and methods, to examine age-related reproductive

performance. Five-week-old Becn1 cKO males showed evidence of mating with females within four days and 71% of the females got pregnant and had a litter (Figure 4.1 J).

Six-week-old Becn1 cKO males had similar performance (Figure 4.1 J); however, 7- and 8-week-old Becn1 cKO males have reduced fertility where less than 50% of plugged females gave birth (Figure 4.1 J). No Becn1 cKO after 9 weeks of age or older sired a litter, despite presence of seminal plugs (Figure 4.1 J). The fertility assays were

completed with a minimum of five Becn1 cKO males for each age tested, demonstrating

149

a fully penetrant phenotype. In contrast, WT mice up to 1-year-old are regularly used in the colony for breeding purposes. Therefore, Becn1 cKO have reduced fertility that quickly diminishes to zero by 9-weeks of age. Thus, Becn1 is required for male fertility after 9 weeks of age.

To determine the basis for reduced fertility, I analyzed reproductive tracts from Becn1 cKO and WT males at several time points from 6- to 17-weeks-old. The body weights of WT and Becn1 cKO showed similar increases with age and there was no difference between genotypes during this period (p>0.5, Holm-Sidak post hoc test) (Figure 4.1 E).

On the other hand, as Becn1 cKO male mice age, their testes undergo regression such that they weigh less than 50% of WT testes by 10-weeks-old (p<0.001, Holm-Sidak post hoc test) (Figure 4.1 F, I). The weight of the seminal vesicles are directly proportional to testosterone production by the Leydig cells of the testes and as such, the weight of the seminal vesicles can be used to monitor testosterone signaling (Deanesly and Parkes 1933; Ayata et al. 1988). The seminal vesicle weights of WT and Becn1 cKO displayed similar increases and were not different between genotypes at any time observed (p>0.2, Holm-Sidak post hoc test) (Figure 4.1 G). This result suggests that Becn1 ablation does not affect Leydig cell function prior to 17-weeks-old and that normal levels of inhibin are being produced by the Sertoli cells. The number of sperm collected from the cauda epididymis of Becn1 cKO mice was markedly reduced compared to WT mice beginning at 8 weeks of age, and this persisted until the end of the study (p<0.001, Holm-Sidak post hoc test) (Figure 4.1 H). At 12, 15 and 17 weeks of age spermatozoa were rarely witnessed in the collection dish, indicating a complete disruption of

spermatogenesis. Moreover, at 6- and 8-week time points, there were sloughed germ cells and leukocytes in the Becn1 cKO collections that were not present in WT

150

collections. Thus, Becn1 is necessary for spermatogenesis and male fertility after 6-weeks-old.