Oct4, Sox2, Klf4, c-My (OSKM) gene therapy in the hypothalamus prolongs fertility and ovulation in female rats

Reproductive aging

In middle-aged female (MA) rats, both the reproductive capacity and frequency of regular estrous cycles are decreased compared to their young adult counterparts [1]. The age-associated changes in estrous cyclicity observed in MA rats are associated to a delay as well as an attenuation of the preovulatory Luteinizing hormone (LH) surge [2], increased LH and follicle-stimulating hormone (FSH) serum levels, and altered circulating estradiol profiles with absence of the surge in the noon of the proestrus with respect to the young rats, [3]. There is clear evidence that in these animals, ovarian failure is preceded by the appearance of a diminished response of the neuroendocrine axis to positive feedback from estradiol [4, 5]. Our group has implemented strategies capable of prolonging reproductive function in female rats. Thus, in female MA rats we have previously demonstrated that intrahypothalamic gene therapy for insulin-like growth factor-I (IGF-I) started at 8 months of age extends the regular cyclicity of the animals beyond 10 months (the age at which MA rats stop ovulating) and preserves the integrity of ovarian structure. At 11 months of age, the treated rats showed, in general, a cyclicity preserved in its regularity as well as a normal ovarian histology, while the controls were, at the same age, mostly acyclic and exhibited a high percentage of polycystic ovaries and few corpora lutea [6].

Aging and epigenetics

The discovery of animal cloning [7, 8] and the subsequent development of cell reprogramming technology by means of the four reprogramming factors, Oct4, Sox2, Klf4, c-Myc (OSKM), also known as the Yamanaka genes, [9] ushered in a technological and conceptual revolution that led to the achievement of cell rejuvenation by full reprogramming and to the emerging view of aging as a reversible epigenetic process where cumulative DNA damage does not appear to play a central role as was thought for decades [10].

Regenerative effects of Yamanaka gene therapy

It is of significant interest that Yamanaka gene therapy in the retina of an experimental-glaucoma mouse model and in middle-aged mice ameliorated their visual acuity [11]. In progeric mice, transgenic for OSKM factors, cyclic partial reprogramming attenuated several signs of aging in visceral tissues and extended by 50% the survival of experimental versus control counterparts [12]. Furthermore, in a recent report [13] it was shown that intravenous gene therapy with a regulatable AAV9 vector system that expresses the OSK genes, implemented in senile mice (29.2 months), prolonged the survival of the animals by two months compared to control counterparts. This is the first report showing that Yamanaka gene therapy in WT mice can extend their survival. We have recently reported that 39-day OSKM gene therapy in the dorsal hippocampus of old rats significantly reverses the typical learning deficits displayed in aged rats [14].

This line of evidence prompted us to implement OSKM gene therapy in the hypothalamus of young female rats using an adenovector constructed by us that expresses a polycistronic cassette (the STEMCCA system) harboring the OSKM genes as well as the GFP gene, all under the control of a bidirectional regulatable promoter [15]. We hypothesized that long-term expression of the OSKM genes in the hypothalamus of young female rats could slow down the rate of decline of fertility in the animals as they near the age of ovulatory cessation (10 months in our rat colony). We report here that 9 months old female rats submitted to hypothalamic OSKM-GFP gene therapy at 4 months of age, display a higher fertility than control vector-treated counterparts and remain cycling regularly at the age of estropause (10 months).

Adenoviral vectors

Construction of a regulatable HD-recombinant adenoviral vector (RAd) Tet-Off adenovector harboring the GFP and Yamanaka genes

The adenovector was constructed using a commercial kit (Microbix Inc., Ontario, Canada) that provides the shuttle plasmid pC4HSU, the helper adenovirus H14 and the HEK293 Cre4 cell line. A full description of the adenovector constructed has been already documented [15]. Briefly, we cloned a construct harboring the bicistronic tandem Oct4-f2A-Klf4-ires-Sox2-p2A-cMyc (known as hSTEMCCA, generously provided by Dr. G. Mostoslavsky, Boston University) under the control of the bidirectional promoter PminCMV-TRE-PminCMV a regulatable Tet-Off promoter which on its second end is flanked by the gene for humanized green fluorescent protein (hGFP).

The hSTEMCCA cassette harbors the 4 Yamanaka genes grouped in pairs placed downstream and upstream of an internal ribosome entry site (IRES). In turn, each pair of genes is separated by a type 2A CHYSEL (cis-acting hydrolase element) self-processing short sequence which causes the ribosome to skip the Gly-Pro bond at the C-terminal end of the 2A sequence thus releasing the peptide upstream the 2A element but continuing with the translation of the downstream mRNA sequence. This allows near stoichiometric co-expression of the two cistrons flanking a 2A-type sequence [16]. The whole expression cassette (STEMCCA cassette 10,065 bp) was cloned into the pC4HSU HD shuttle at the AscI and SwaI sites giving rise to pC4HSU-STEMCCA-tTA (Figure 1). The pC4HSU HD shuttle consists of the inverted terminal repeats (ITRs) for Ad 5 virus, the packaging signal and part of the E4 adenoviral region plus a stuffer non-coding DNA of human origin which keeps a suitable size (28–31 Kbp) of the viral DNA.

Figure 1. Diagrammatic representation of main intermediate constructs generated during the construction of HD-RAd-STEMCCA-GFP-Tet-Off. (A) Shows the base plasmid, pLPBL-BD-tTA-GFP, from which we departed in the generation of the above adenovector. It harbors the CMV1-TRE-CMV2 regulatable promoter flanked on one side by the gene for GFP and by a multiple cloning site (MCS) on the other side. Separately, the plasmid also harbors the gene for the chimeric tetracycline transactivator (tTA) protein, created by fusing one protein, TetR (tetracycline repressor), with the activation domain VP16, from the Herpes Simplex Virus. This construct is under the control of the human cytomegalovirus (CMV) promoter. (B) Displays the above plasmid now harboring the STEMCCA tandem, which was cloned into the MSC site. (C) Shows the full STEMCCA construct (including all associated regulatory components) cloned into the HD genomic plasmid which includes a bacterial sequence between its ITRs, flanked by PmeI sites. (D) Illustrates the basic components of HD-RAd-STEMCCA-GFP-Tet-Off genome after removing the bacterial sequence from pC4HSU-tTA-STEMCCA-GFP. For further details see Figure 2A legend and Ref. 15.4.

The linearized DNA backbone of the new HD-RAd (Figure 2A) was transfected in Cre 293 cells. For expansion, the helper H14 adenovirus was added to the cell cultures at a multiplicity of infection (MOI) of 5. In H14, the packaging signal is flanked by lox P sites recognized by the Cre recombinase expressed by the 293 Cre4 cells. Therefore, the helper virus provides in trans all of the viral products necessary for generation of the desired HD-RAd. The infected 293 Cre4 cells were left for 2–3 days until cytopathic effect (CPE) was evident. Cells and medium were collected and submitted to 3 freeze-thaw cycles to lyse them. Clear lysates were obtained, mixed with H14 helper virus and added to a fresh culture of Cre4 293 cells at MOI 1. When CPE appeared, passage 2 (P2) cell lysates were prepared. This iterative co-infection process was carried on five more times in order to generate enough HD-RAd particles as to proceed to the purification step. The newly generated HD-RAd was rescued from P5. The generated HD-RAd was purified by ultracentrifugation in CsCl gradients. Final virus stock was titrated by lysing the viral particles, extracting their DNA and determining its concentration in a Nanodrop spectrophotometer. For the first preparation, titer was 12 × 10¹¹ physical viral particles (pvp)/ml.

Figure 2. (A) The figure illustrates the basic components of HD-RAd-STEMCCA-GFP-Tet-Off genome. Abbreviations: GFP: humanized Green Fluorescent Protein; TRE: Tetracycline responsive element; tTA: chimeric regulatory protein; PminCMV: cytomegalovirus minimal promoter; SV40pA: polyadenylation signal; ITR: inverted terminal repeats; ψ: packaging signal. (B) Expression of GFP from the adenovector HD-RAd-STEMCCA-GFP in the third ventricle of the rat hypothalamus 6 days after vector injection in the lateral ventricles. Notice the processes of the ependymal tanycytes forming an extensive network in the hypothalamic parenchyma. Scale bar, 50 µm (for both, B, C). (C) Expression of DsRed2 from the control adenovector HD-RAd-DsRed2 in the rat hypothalamus 30 days after vector injection. Notice the processes of the ependymal tanycytes forming an extensive network in the hypothalamic parenchyma.

We also constructed two control regulatable HD-RAd Tet-Off harboring either the hGFP gene or the gene for the DsRed2, red fluorescent protein isolated from Discosoma sp.

Animals and in vivo procedures

Female Sprague-Dawley rats aged 3–4, 10 and 26 months were used. The animals were raised in our institution (INIBIOLP) and housed in a temperature-controlled room (22 ± 2ºC) on a 12:12 h light/dark cycle (lights on from 7 to 19 o’clock). Food and water were available ad libitum. All experiments with animals were performed according to the Animal Welfare Guidelines of NIH (INIBIOLP’s Animal Welfare Assurance No A5647-01).

Assessment of the effect of age on cycle regularity

Females aged 3, 10 and 26 months were submitted to daily vaginal smears sampling for 30 days.

Hypothalamic section assessment by fluorescence microscopy

Some of the rats were used for immunofluorescence studies in the hypothalamus. Animals were placed under deep anesthesia and perfused with phosphate buffered formaldehyde 4%, (pH 7.4) fixative. Each brain was removed and trimmed down to a block containing the whole hypothalamus. The block was then serially cut into coronal sections 40 µm thick on a vibratome (Leica, Nussloch, Germany).

Hypothalamic brain sections were mounted with Fluoromount water-soluble mounting medium. Images of hypothalamic and other brain sections were captured using a confocal Olympus FV1000 microscope (Tokyo, Japan). Digital images were analyzed using the ImagePro Plus (IPP^™) v6.3.

Statistical analysis

The t-test was used to evaluate group differences.

HD-RAd OSKM-GFP expression in the hypothalamus of young rats

Both, the HD-RAd OSKM-GFP and the control HD-RAd vectors were stereotaxically injected in the MBH of young female rats. Six days after injection, hGFP or DsRed expression was strong. The processes of the ependymal tanycytes form an extensive network in the hypothalamic parenchyma (Figure 2B). When our control vector HD-RAd-DsRed2 was injected into the hypothalamic parenchyma of young rats, DsRed 2 expression remained high for at least 30 days (Figure 2C).

Characterization of estrous cycle patterns throughout the lifespan

Using intact animals from our female rat colony, we performed an initial characterization of estrous cycle patterns from youth through very old age. As expected, the results showed that transition from regular to irregular cyclicity takes place at around 9 months of age and is followed by a prevalence of CE status from age 10 to 18–20 months (Figure 3). Animals older than 20 months progressively go into a constant diestrus phase. Thus, 3-month-old females typically show 4–5-day estrous cycles characterized by one day or less of P (in some cycles P was so short that when vaginal smear was performed the cell proportion was consistent with proestrus entering estrus or sometimes just estrus), one-two days of E and 2–3 days of D1 and/or D2. Ten-month old females typically show a prevalence of lengthy CE periods with interspersed irregular cycles (Figure 3, middle panel). Their ovarian histology showed numerous follicular cysts and scarce CL (data not shown, see ref 6). In senescent females (26 months) CE gave way to an increasing incidence of long periods of D with occasional 2–3 day-periods of P and E vaginal cytology as well as some CE periods. Senescent ovaries showed follicular cysts and some CL (data not shown).

Figure 3. Estrous cycle patterns in young (3 momths, A), middle-aged (10 months, B) and old (26 months, C) female rats. The Y axis represents the estrous cycle phases of the animal on the indicated day of a 31-day window (X-axis) as determined by vaginal cytology. Four estrous phases were defined according to the proportion of epithelial and cornified cells or leukocytes; Proestrus (P) was characterized by abundance of epithelial cells and the presence of leukocytes. Estrus (E), showed abundance of cornified cells and to a lesser extent epithelial cells. Diestrus 1 (D1), was defined by the presence of an increasing number of leukocytes accompanied by substantial numbers of cornified cells. Diestrus 2 (D2) was characterized by high abundance of leukocyes which constituted the vast majority of cells present in the vaginal smears at D2.

Effect of hypothalamic OSKM gene therapy on fertility in MA female rats

As expected, the young group showed a much higher pregnancy rate (83%) than the MA controls (8.3%). Interestingly, MA OSKM treated rats showed a 25% rate of fertility, which although far lower than in young rats, was 3 times higher than in MA controls. (Figure 4B). When a z-test for proportions was applied to 8.3 vs. 25%, the two-sided p-value was 0.13104, while the one-sided p-value was 0.06552. Since the null hypothesis is one-sided, the one-sided value was used in our analysis.

Figure 4. Effect of long-term OSKM-GFP gene therapy on the pregnancy rate, litter size, pup BW and estrous cyclicity in MA female rats. (A) There were three experimental groups; 4 months old intact females (Young, N = 12), 9.3 months old control rats (MA control N = 12) and 9.3 months old OSKM-treated rats (MA OSKM N = 12). These N values are indicated on each bar. (B) Pups were weighed when they were 1 or 2 days old and again at weaning (21 days). (C) During the 10th month of life daily vaginal smears were taken in the OSKM-treated rats in order to assess the effect of the OSKM gene therapy on regularity of estrous cyclicity. The estrous cycle phases are indicated as follows. Abbreviations: P: Proestrus; E: Estrus; D1: Early diestrus; D2: Late diestrus.

Also as expected, the litter size was higher in the young (mean litter size 9.1 pups) than in the MA controls (Figure 4B, inset) and OSKM-treated (mean litter size, 3 pups in each group). The body weight of the pups at birth was lower in the young rats (mean BW in pups from young rats, 9.2 g) than in the MA controls and treated (mean BW, 11.2 and 11.8 g, respectively). (Figure 4A). However, at weaning the pups of the OSKM animals had gained more weight than the pups of the MA-C females (Figure 4A). The BW of the pups from the young females remained lower than the OSKM counterparts, a fact that may be accounted for by the higher number of pups the young mothers had to feed as compared with the OSKM MA rats (8 vs. 3, respectively). In the case of the pups from the MA-control females, the lower BW at weaning suggests that milk production and/or quality declines with age, a change that was attenuated by hypothalamic OSKM gene therapy.

The total number of pups born from Y, MA-C and MA-OSKM was 83, 3 and 9, respectively. All pups survived and showed similarly normal behavior in the three groups.

Interestingly, most OSKM-treated MA rats were still cycling regularly at age 10 months (Figure 4C), which is in contrast with what happened in 10-month-old MA intact controls which showed irregular cycles with the prevalence of CE status (Figure 3, middle panel).