S. Yao

WEE2 Is an Oocyte-Specific Meiosis Inhibitor in Rhesus Macaque Monkeys

WEE1 homolog 2 (WEE2, also known as WEE1B) is a newly identified member of the WEE kinase family that is conserved from yeast to humans. The aim of the present study was to determine the spatiotemporal expression pattern and the function of WEE2 during oocyte maturation in a nonhuman primate species, the rhesus macaque. Among 11 macaque tissues examined, WEE2 transcript is predominantly expressed in the ovary and only weakly detectable in the testis. Within the ovary, WEE2 mRNA is exclusively localized in the oocyte and appears to accumulate during folliculogenesis, reaching the highest level in preovulatory follicles. Microinjection of a full-length WEE2-GFP (green fluorescent protein) fusion mRNA indicates a specific nuclear localization of WEE2 protein in both growing and fully grown germinal vesicle (GV)-intact oocytes. Taking the long double-stranded RNA-mediated RNA interference approach, we found that down-regulation of WEE2 led to meiotic resumption in a subset of GV oocytes even in the presence of a phosphodiesterase 3 inhibitor. On the other hand, overexpression of WEE2 delays the reentry of oocytes into meiosis in both mice and monkeys. These findings suggest that WEE2 is a conserved oocyte-specific meiosis inhibitor that functions downstream of cAMP in nonhuman primates.

Blockade of tubal patency following transcervical administration of polidocanol foam: initial studies in rhesus macaques ☆ ☆☆

OBJECTIVE To demonstrate the feasibility of polidocanol foam (PF) as a nonsurgical method of female permanent contraception using a nonhuman primate model. STUDY DESIGN Four groups of adult female rhesus macaques underwent either transcervical treatment with 5% PF directly into the uterine cavity, treatment with inert (methylcellulose, MF) foam or no treatment followed by removal of the reproductive tract for histologic evaluation. Untreated animals were included in Group 1 (n=3). Group 2 animals (n=4) were treated once with MF. Group 3 (n=7) received a single, and Group 4 (n=5) received multiple monthly treatments with PF; in these 2 groups, baseline tubal patency was assessed either laparoscopically by chromopertubation (CP) or by hysterosalpingography. RESULTS Group 1 (untreated) and Group 2 (MF) animals had normal tubal histology. In contrast, Group 3 and 4 females treated with PF showed evidence of tubal damage. In Group 4, bilateral tubal blockade was noted on CP after two (n=2) or three (n=3) treatments. Histologic analysis confirmed complete tubal occlusion (loss of epithelium, fibrosis) in three of these animals, and one showed significant tubal damage localized to the intramural segment. Nontarget (cervix, vagina, endometrium, ovary) reproductive tissues were unaffected. While similar tubal changes were observed after a single treatment (Group 3), endometrial hemorrhage was also noted as an acute change. CONCLUSION PF is a promising candidate agent for nonsurgical permanent female contraception. The histologic features of PF occlusion are confined to the intramural portion of the tube. IMPLICATIONS This study in rhesus macaques supports further development of transcervical administration of PF as a nonsurgical approach to permanent contraception. A nonsurgical method could reduce risks and costs associated with surgical female sterilization and increase access to permanent contraception.

Identification of phosphodiesterase 9A as a cyclic guanosine monophosphate–specific phosphodiesterase in germinal vesicle oocytes: a proposed role in the resumption of meiosis

OBJECTIVE To identify a cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase (PDE) in nonhuman primate germinal vesicle (GV) oocytes and establish a proposed effect on oocyte maturation through preliminary experiments in mouse GV oocytes. DESIGN Controlled nonhuman primate and rodent experiments. SETTING Academic research institution. ANIMAL(S) Rhesus macaques and B6/129F1 mice. INTERVENTION(S) Stimulation of Rhesus macaques with follicle-stimulating hormone (FSH) to collect GV oocytes and cumulus for gene expression analysis, and stimulation of female mice with pregnant mare serum gonadotropin to collect GV oocytes. MAIN OUTCOME MEASURE(S) Expression of PDE transcript in primate GV oocytes and cumulus cells, measurement of fluorescence polarization of phosphodiesterase 3A (PDE3A) activity, and analysis of spontaneous resumption of meiosis in mouse GV oocytes. RESULT(S) Of five PDE transcripts detected in Rhesus GV oocytes, only PDE9A was cGMP-specific. The fluorescence polarization assays indicated cGMP has an inhibitory effect on PDE3A while the phosphodiesterase 9A (PDE9) inhibitor, BAY73-6691, does not. Similarly, BAY73-6691 had little effect on preventing spontaneous maturation in oocytes, but did augment the inhibitory effects of cGMP. Inclusion of 0 μM (control), 10 μM, 100 μM, and 1 mM BAY73-6691 statistically significantly increased the proportion of mouse oocytes maintaining GV arrest in the presence of the cGMP analog 8-Br-cGMP at 100 μM (8.8%, 11.4%, 18.8%, and 28%), 500 μM (21.1%, 38.1%, 74.5%, and 66.5%), and 1 mM (57.8%, 74.5%, 93.9%, and 94.0%), respectively. CONCLUSION(S) Phosphodiesterase 9A (PDE9A) is a cGMP-specific hydrolyzing enzyme present in primate oocytes, and PDE9 antagonists augment the inhibitory effect of cGMP during spontaneous in vitro maturation of GV mouse oocytes.

Characterization of tubal occlusion after transcervical polidocanol foam (PF) infusion in baboons

OBJECTIVE Our long-term goal is to develop a nonsurgical method of fallopian tubal occlusion for the purpose of permanent contraception. We have previously demonstrated that transcervical administration of 5% polidocanol foam (PF) can create tubal occlusion in macaques but that multiple treatments are required. In this study, we assessed the efficacy of various regimens of PF with and without depomedroxyprogesterone acetate (DMPA) (to control ovarian cycle phase) in the baboon. STUDY DESIGN Adult cycling female baboons were evaluated for tubal patency by hysterosalpingography and then received a transcervical infusion of PF with (+) or without (-) an intramuscular injection of DMPA (3.5 mg/kg). Two concentrations of PF were compared: 1% [(+) DMPA, n=5; (-) DMPA, n=3] and 5% [(+) DMPA, n=4; (-) DMPA, n=3]. Controls received (+) DMPA (n=2) or (-) DMPA, (n=3) only. The reproductive tracts were removed 1-3 months after treatment for examination. RESULTS No fallopian tubal occlusion was observed in negative controls (±DMPA). Histologic complete tubal occlusion was observed in 3/8 of females treated with 1% PF and in 6/7 treated with 5% PF. Histologic evaluation suggested that 1% PF is associated with prolonged chronic inflammation (more than 2-3 months), while 5% treatment eliminates the epithelial lining, at least focally, and resolves into complete occlusion within 1-2 months. This pattern of complete occlusion was seen in all 4 females that received 5% PF (+DMPA) and in 2/3 that received 5% PF (-DMPA). CONCLUSION In a baboon model of transcervical permanent contraception, a single treatment with 5% PF resulted in complete tubal occlusion more reliably (85%) than 1% PF (38%). Cotreatment with DMPA may improve treatment results with 5% PF but requires additional study. IMPLICATIONS A finding that a single transcervical treatment with 5% PF can occlude the fallopian tubes of baboon supports further study of this approach as a novel strategy for permanent contraception for women.

Transcervical administration of polidocanol foam prevents pregnancy in female baboons

Background Our objective was to conduct a pilot study to determine if transcervical administration of polidocanol foam (PF) with or without doxycycline or benzalkonium chloride (BZK) would prevent pregnancy in baboons. Methods In study phase 1, adult cycling baboons underwent a hysterosalpingogram to evaluate tubal patency prior to transcervical infusion of 20 mL of 5% PF followed by 1 mL of saline containing 100 mg doxycycline (5%/doxy; n=5), 3% PF plus doxycycline (3%/doxy; n=4), 3% PF with 0.01% BZK (3%/BZK; n=4) or no additional treatment (control; n=9). Immediately following treatment, animals received intramuscular depot medroxyprogesterone acetate (DMPA, 2 mg/kg) to suppress cyclicity during healing and were then socially housed with males of proven fertility. The primary outcome was pregnancy within six cycles of resumption of menses (efficacy phase 1). During study phase 2, PF-treated females from study phase 1 contributed additional cycles (6–8) of exposure (efficacy phase 2), and 5 control females who had recovered from medical abortion (after study phase 1 pregnancy) were subsequently treated with 5% PF (with DMPA) and exposed to breeding (efficacy phase 1; n=3 six cycles, n=2 five cycles). Results All females resumed normal menstrual cycles and mating activity after DMPA. During efficacy phase 1, 7/9 (78%) control females became pregnant. In contrast, fewer pregnancies occurred in PF-treated females: 5% PF 0/5 (0%), 5%/doxy 1/5 (20%), 3%/doxy 1/4 (25%) and 3%/BZK 1/4 (25%). During efficacy phase 2, only one additional pregnancy occurred (3%/BZK). Conclusions A single transcervical treatment with 5% PF prevented pregnancy in most baboons. Cotreatment with doxycycline or BZK did not improve results. Implications Transcervical intrauterine administration of PF resulted in a high rate of tubal occlusion with prevention of pregnancy; refinements are needed to increase the contraceptive rate following a single treatment to near 100%.

Phosphodiesterase 3 (PDE3) inhibition with cilostazol does not block in vivo oocyte maturation in rhesus macaques (Macaca mulatta).

OBJECTIVE Studies in mice suggest that cilostazol, an FDA-approved phosphodiesterase 3 (PDE3) inhibitor, might have a contraceptive effect within the approved dose range. We sought to evaluate the potential contraceptive effects of cilostazol in a nonhuman primate model. STUDY DESIGN Adult female rhesus macaques were stimulated to develop multiple preovulatory follicles by administering human recombinant gonadotropins, and oocytes were collected by follicle aspiration 36 h after an ovulatory stimulus (human chorionic gonadotropin). Monkeys received no further treatment (controls) or the PDE3 inhibitor cilostazol at the maximum approved human dose of 100mg twice daily starting 6 days prior to follicle aspiration. Recovered oocytes were scored for meiotic stage [germinal vesicle (GV) intact, GV breakdown], and metaphase II stage oocytes were fertilized in vitro and observed for normal embryo development. RESULTS Similar proportions of GV stage oocytes were recovered from control (27%±4%) and cilostazol (27%±9%)-treated females, and the proportion of embryos that developed into blastocysts was also similar for both groups (7%±5% control vs. 15%±8% cilostazol). CONCLUSION Oral dosing of cilostazol tablets during controlled ovarian stimulation protocols did not prevent oocyte maturation or embryo development in macaques. IMPLICATIONS Since administration of the maximum approved human dose of cilostazol (an FDA-approved PDE3 inhibitor) to macaques did not prevent oocyte maturation or fertilization, it is not likely that this dose would be contraceptive in women.

Polidocanol induced tubal occlusion in nonhuman primates: immunohistochemical detection of collagen I-V

Objective Intrauterine administration of polidocanol foam (PF) can create fallopian tube occlusion in nonhuman primates. The objective of this study was to determine if PF-induced tubal obstructions contain collagen in the extracellular matrix. Study design We compared tissue samples of the intramural fallopian tube obtained from previous studies evaluating the effects of intrauterine infusion of 5% PF 2–12 weeks after treatment. Serial sections of the intramural portion of the fallopian tube obtained from representative treated (rhesus macaques, n=7; baboon, n=11) and untreated control (macaque, n=3; baboon, n=5) animals were stained with hematoxylin and eosin to identify tubal occlusion and by immunohistochemistry for collagens Col-I, Col-III and Col-IV. Descriptive results are summarized. Results Control animals exhibited histologically normal fallopian tubal epithelium with no staining for Col-1, light staining for Col-III and Col-V in the lamina propria and Col-IV distributed evenly in the extracellular matrix of the lamina propria. Treatment with PF resulted in acute tissue damage confined to the intramural tube; no epithelial damage or occlusion occurred in the tubal isthmus or ampulla. Blockade of the intramural tube demonstrated fibrosis with the epithelium replaced with extracellular matrix that stained strongly for Col-I, Col-III, Col-IV and Col-V. Col-II was undetectable. Conclusion Tubal blockage induced by PF resulted in loss of normal epithelium and accumulation of collagens Col-I, Col-III, Col-IV and Col-V at the site of obstruction. The presence of dense collagen staining supports the hypothesis that PF infusion creates lasting tubal obstructions. Implications This study demonstrates that PF-induced tubal occlusion results in deposition of collagens suggesting the potential for a more lasting blockade. The structural nature of this occlusion supports the development of intrauterine administration of PF as a nonsurgical method of permanent contraception.