Pregnancy and live birth from nonsurgical transfer of in vivo- and in vitro-produced blastocysts in the rhesus monkey

Embryo transfer in the rhesus monkey has been historically limited to transfer of cleavage stage embryos. In order to allow genetic manipulation of rhesus embryos in vitro, without using invasive surgical techniques, it is important to explore the transfer of morula and blastocyst stage embryos. Embryos were produced by in vitro fertilization from gonadotropin-stimulated monkeys, or were obtained by nonsurgical uterine flushing of naturally mated or artificially inseminated females. Nonsurgical transfer was accomplished by inserting a metal guide through the cervix into the uterus, after which a hollow cell sampler was inserted over the guide. The guide was removed and a catheter was inserted containing one to five embryos. Several pregnancies resulted from in vitro- and in vivo-derived blastocysts, and two pregnancies were carried to term resulting in one live birth. Blood samples were collected regularly to monitor plasma levels of chorionic gonadotropin, luteinizing hormone, and progesterone. The recipients received progesterone as a subcutaneous implant or daily injections from the day of transfer. The approach described in this study provides the opportunity to explore transgenic and chimeric models in the monkey by the development of noninvasive methods to transfer late-stage embryos that have been manipulated in vitro.     

Successful recovery of preimplantation embryos by nonsurgical uterine flushing in the bonnet monkey

A major limitation to progress in primate embryology is the lack of an adequate supply of preimplantation embryos. We describe a method for recovering preimplantation-embryos in bonnet monkeys (Macaca radiata ) using a nonsurgical uterine flushing technique similar to the one previously employed in rhesus monkeys. Forty cyclic females were screened for cervical cannulation, and 10% of these had an impassable cervix. Eleven females suitable for cannulation were selected, and 27 menstrual cycles were monitored over a 5-mo period. Seventy-one percent of the cycles showed estrogen peaks, which were observed between Days 9 and 14 of the cycle. Following natural mating, uterine flushings were performed on Days 5 to 8 of pregnancy (Day 0 = the day following the estrogen peak). Of the 27 recovery attempts, 9 (33.3%) resulted in the recovery of ovulation products, including those of an unfertilized oocyte and empty zona (2 cases), retarded cleavage-stage (4 to 8-cell) embryos (4 cases), morula (1 case) and blastocysts (2 cases). These results show, for the first time, that the nonsurgical uterine flushing technique can be successfully performed to recover uterine-stage preimplantation embryos from bonnet monkeys.     

恒河猴正常子宫及妊娠后期胎儿的MRI研究

目的研究恒河猴正常子宫及妊娠后期胎儿的MRI表现。方法对3只未妊娠和3只妊娠135d的恒河猴分别进行磁共振成像（MRI）扫描,观察子宫及胎儿的影像学特点。结果未妊娠恒河猴的子宫T1WI冠状位呈椭圆形,子宫体各层呈中等信号,矢状位呈葫芦形。在T2WI上,冠状位显示宫体可见2～3层不同的信号带,在矢状位子宫体肌层的信号高于子宫颈,信号的移行区是体颈的交界处。妊娠恒河猴的子宫肌层变薄,胎盘及胎儿的脑、脊柱、肝、肺等结构显示清楚。结论MRI能很好地显示恒河猴子宫的形态、胎儿各部分的结构。对人畸形胎儿的产前诊断有一定的参考价值。     

Radioligand binding studies reveal marked species differences in the vasopressin V1 receptor of rat, rhesus and human tissues.

The [3H]arginine-vasopressin ([3H]AVP) binding site in rat, rhesus and human liver and nonpregnant human uterus was characterized and contrasted. [3H]AVP bound with high affinity (Ki values, 0.2-0.6 nM) to preparations of all tissues studied. Competition binding studies using a series of compounds from three structural classes indicate a marked species difference between the rat and primate liver AVP-V1 site. This site in rhesus and human liver however, is essentially identical, indicating that the rhesus liver is an appropriate surrogate for human tissue. These studies also indicate that the AVP-V1 site of nonpregnant human uterus and human liver is equivalent.     

Effect of altered hormonal states on transaminase enzymes in the genital ograns of immature female rhesus monkeys

The effect of ovariectomy and hormone replacement therapy on the activity of glutamate oxalacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) in the oviduct, uterus, cervix and vagina of immature female rhesus monkeys (Macaca mulatta ) was studied. In intact animals, GOT activity was high in the oviduct, whereas GPT was high in the vagina. Ovariectomy suppressed the activity of both the enzymes in varying degrees. Estradiol dipropionate stimulated GOT in the oviduct and uterus, whereas progesterone increased this enzyme in the uterus. Sequential treatment of two hormones inhibited the enzymes in all the tissues; GPT was, however, stimulated in the fundus region of the uterus. The study demonstrated the presence of transaminase enzymes in the genital tissues of rhesus monkey and their differential response to sex steroids.     

Nonsurgical collection of embryos in Shiba goats

A method of nonsurgical embryo collection in the Shiba goat, a native Japanese miniature goat breeding nonseasonally, was developed. The apparatus used for flushing the uterus was made on the model of the two-way catheter for cows. Embryo collection was performed on days 5 to 7 in 37 females superovulated with PMSG and hCG and resulted in successful recovery of 69 embryos in 19 females (51.4%). The average number of embryos collected from each successful female was 3.6. The recovery rate of embryos calculated on the basis of the number of embryos recovered and corpora lutea observed by culdoscopy in 15 successful females was 89.5%. This nonsurgical method seem to be efficient enough for collecting morulae and blastocysts in Shiba goats.     

一种非手术性小鼠胚胎移植技术

为了提高非手术性小鼠胚胎移植技术的成功率,利用塑料移植导管模拟非手术胚胎移植过程,通过观察染料在子宫角的分布而评估胚胎移植效果,并在此基础上将自然妊娠3.5 d的小鼠囊胚经子宫颈移植受体小鼠。结果表明:将CD-1小鼠囊胚移植假孕2.5 d小鼠单侧子宫角,平均70.9%的胚胎能够发育至成活新生仔鼠,建立了高效非手术性小鼠胚胎移植技术。该方法简便快捷、不易污染、费用低,无需专业的手术器械,且符合实验动物伦理原则,完全可以取代手术法胚胎移植技术,更重要的是,它为人类和其他大动物的胚胎移植提供了研究模型。     

Cytoskeletal remodelling proteins identified in fetal-maternal interface in pregnant women and rhesus monkeys

The uterus undergoes dramatic remodelling in preparation for embryo implantation and pregnancy establishment. A receptive uterus is pivotal for embryo attachment, implantation and the eventual formation of a hemochorial placenta. We have previously identified by proteomics that tropomyosin alpha-4 chain (TPM4), protein disulfide isomerase A1 (PDIA1) and src substrate cortactin 8 (SRC8) were up regulated in the decidualized stromal cells during the late secretory phase of the menstrual cycle in women. These three proteins are associated with cytoskeletal remodelling. This study determined the localization of these three cytoskeletal proteins in the fetal-maternal interface including the decidual cells in the 1st trimester of pregnancy in women and rhesus monkeys. Immunohistochemical analysis revealed that TPM4, PDIA1 and SRC8 were all expressed by the decidual cells and the wall of the spiral arterioles in pregnant women. Similar expression pattern were also found in the rhesus monkey. In addition, TPM4, PDIA and SRC8 were also localized to the trophoblast cells, further highlighting the importance of these cytoskeletal remodelling proteins in early pregnancy.     

A single cannula technique for nonsurgical collection of ova from cattle.

The uteri of 34 heifers were flushed for ova six to nine days following estrus using a single cannula nonsurgical technique. The technique involved the infusion of fluid by gravity and agitation within the uterus by to-and-fro action of a syringe followed by unassisted fluid collection. Each horn was flushed five times using 30–150 ml of flushing fluid per flush. Recovered fluid (flushing fluid plus uterine secretion) was an average of 95% of the volume of the fluid inserted. Ova were recovered from 12 of 19 nontreated, single ovulating heifers (63%) and from all of 15 superovulated heifers (mean and S.D. for number of ova, 6.3 ± 4.4/ superovulated heifer; range, 1 to 14 ova). Based on the number of corpora lutea, the ova recovery index was 54% as averaged over the 15 superovulated heifers. The technique has been used in 4 additional superovulated heifers with modification (increased number of flushes to 8) subsequent to the termination of the planned project. Recovery index for the first 5 flushes was 58%. However, some ova were recovered in the 6th, 7th, and 8th flushes resulting in an apparent improved recovery index of 69%.     

Concanavalin a capping of rhesus monkey polymorphonuclear leukocytes

The technique of capping, a probe designed to evaluate the fluidity and functional competence of human polymorphonuclear leukocytes (PMNs), has been successfully adapted for rhesus monkey PMNs. The capping characteristics of rhesus monkey PMNs are very similar to those of human PMNs. Utilizing this capping technique as an evaluation tool and with fetal/neonatal rhesus monkey as a functional animal model, the ontogeny of movement and chemotactic characteristics of PMNs can now be studied.     

Successful culmination of pregnancy and live birth following the transfer of frozen-thawed buffalo embryos

A total of 141 embryos was recovered by nonsurgical flushing of the uterus of 31 superovulated buffalo. A total of 66 good quality embryos (Grade I and Grade II) was frozen using 1.4 M glycerol. Forty-two of the frozen embryos were thawed randomly over a 1-year period, and a total of 39 embryos (Grades I, II or III post thaw) were transferred into an equal number of estrus synchronized recipients. Of 11 confirmed pregnancies, 9 calves were born live.     

Evaluation of ultrasonography for monitoring follicular growth in rhesus monkeys

The purpose of this study was to determine if the ovaries and uterus of rhesus monkeys could be visualized by ultrasonography and to detect changes associated with follicular growth and ovulation. Animals were examined during 15 menstrual cycles, for an average of nine consecutive days. Ultrasonic recordings were correlated with hormonal parameters (estradiol 17beta, E(2); luteinizing hormone, LH; and progesterone, P) and laparoscopic findings. The uterus and both ovaries were observed in more than 90% of the examinations. A dominant follicle (DF) was identified during all ovulatory cycles, on average 1 d preceding the E(2) peak. The maximal diameter of the DF ranged from 3 to 7 mm. Laparoscopic examinations to determine the site of the DF confirmed ultrasonic findings in 10 of 14 cycles (P < 0.1). There was no significant difference in the size of the dominant and contralateral ovaries; however, more follicles with a diameter of 2 to 7 mm were found on the dominant ovary (P < 0.05). Two animals stimulated with exogenous gonadotropins showed a linear increase in ovarian size for 6 d prior to oocyte recovery (P < 0.05), reflecting an increase in the number of developing follicles. Ultrasonography can be used to identify the DF during spontaneous cycles in rhesus monkeys and to monitor the response of monkeys to exogenous gonadotropins.     

Role of progesterone on peri-implantation stage endometrium-embryo interaction in the primate

Progesterone secretion during the luteal phase influences oviductal and endometrial functions which are essential for embryo viability and implantation in a number of species including primates. Luteal phase estrogen is not essential for progesterone-dependent endometrial receptivity towards implantation and pregnancy in the rhesus monkey and in the human. However, synchronous development of embryo and endometrium is an essential prerequisite for evolutive implantation. Progesterone helps to maintain synchronous development of preimplantation embryo through its action on maternal uterus. The anti-nidatory action of mifepristone, a potent progesterone receptor modulator (PRM) with pronounced antiprogestagenic activity, is known to be associated with desynchronization of endometrium along with repression of glandular secretory differentiation and vascular maturation. Thus, it is likely that early luteal phase administration of mifepristone affects paracrine action of the secretory stage endometrium on the preimplantation stage embryo, and thereby inhibits embryonic development and viability. We shall examine this hypothesis using the rhesus monkey as a primate model.     

Estrogen interconversions in the induced cycle in female rhesus monkeys

To determine the extractions and interconversions of estrone and estradiol across and within the uterus, [3H]estradiol and [14C]estrone were infused at a constant rate in six ovariectomized female rhesus (Macaca mulatta) monkeys. Studies were done on Days 9, 14, and 23 of artificial menstrual cycles induced by the timed insertion and removal of Silastic capsules of estradiol and progesterone. Measurements of estrogen radioactivity were made from peripheral arterial blood and uterine venous blood as well as from endometrial biopsy samples. A significant increase occurred in the conversion of estradiol to estrone measured within the uterus on Day 23 compared to Days 9 and 14. The conversion of estrone to estradiol, measured within the uterus, fell progressively from Day 9 to Day 23, but this decrease was not significant. The extractions and interconversions across the uterus, and the overall interconversions of estrone and estradiol were not significantly different on Days 9, 14, or 23 of the cycle. Thus, we have been able to confirm in vivo the increase in the activity of the 17 beta-hydroxysteroid dehydrogenase, the enzyme responsible for estradiol to estrone interconversions, shown earlier by studies done in vitro. However, the increase in 17 beta-hydroxysteroid activity in the uterus is not reflected in the overall interconversions of estrone and estradiol as reflected by measurements in peripheral arterial blood.     

Fluorescence in situ hybridization using an old world monkey Y chromosome specific probe combined with immunofluorescence staining on rhesus monkey tissues.

To date, there is no commercially available Y chromosome probe that can be used for fluorescence in situ hybridization (FISH) for the male rhesus monkey. We have recently generated a probe for FISH with high specificity to the short arm of the rhesus monkey Y chromosome. In this study, we further describe a method that keeps the integrity of tissue-specific antigenic structures for immunofluorescence staining subsequent to FISH on paraffin-embedded rhesus monkey tissues. We have examined this technique in combination with an epithelial cell-specific marker, cytokeratin 8/18 (CK8/18), on various tissues, including jejunum, liver, kidney, and pancreas. CK8/18 and Y chromosome signals were distinctly seen simultaneously on epithelial cells from the same tissue section from male but not female monkeys. These studies indicate that our FISH immunofluorescence technique can be reliably used to identify and phenotype male cells in paraffin-embedded rhesus monkey tissues.     

Magnetic resonance imaging of the placenta in rhesus monkeys, Macaca mulatta

Magnetic resonance imaging of the uterus of rhesus monkeys was performed at different stages of pregnancy. Spin-echo sequences localized the placental discs before and after gadolinium enhancement. Fast-scan sequences provided semiquantitative appreciation of placental hemodynamics. The paramagnetic agent enhanced the placenta, crossed into the fetal circulation, and was excreted in the fetal urinary bladder. Gd DTPA was also used to contrast the external contours of the fetus.     

A microagglutination test for blood typing rhesus monkeys, Macaca mulatta

Summary. We have developed a microagglutination test for typing rhesus monkey erythrocytes that is sensitive, accurate and easy to perform. The technique requires only μ1 quantities of antiserum and cells, and agglutination is easily detected using an inverted microscope. An advantage of this technique is that the typing plates can be stored at -70°C without loss of activity. The results of typing over 400 rhesus blood samples with this technique were 95% concordant with results using the standard microtitre agglutination technique. Preliminary results indicate that this test is also adaptable to typing human blood.     

Stromal decidualization of endometriosis in the rhesus macaque (Macaca mulatta): a case report

During exploratory laparotomy, a 10-year-old female rhesus macaque was found to have a 6.0 x 9.5 x 2.0-cm multichambered, yellow, cystic mass cranial to the uterus, from which large amounts of opaque, white fluid were discharged into the abdominal cavity. The animal was euthanized, and the body was submitted for gross and histologic evaluation. Sections of the mass examined microscopically consisted of sheets of polygonal to round cells, with well defined cell borders and moderate amounts of eosinophilic cytoplasm. Scattered throughout these cells were few, variably sized glandular structures composed of columnar to cuboidal epithelium. Glandular epithelial cells were positive for keratin, and the sheets of polygonal cells were positive for vimentin and negative for keratin and CD 68. Gross and histologic appearance, immunohistochemical findings, and history of medroxyprogesterone acetate injections were compatible with a diagnosis of stromal decidualization of endometriosis. Subsequent biopsies of similar lesions in other rhesus macaques in the colony being treated with medroxyprogesterone acetate for endometriosis revealed comparable histologic findings.     

恒河猴群微卫星DNA多态性的分析

目的　确立一种对恒河猴群个体的遗传物质进行准确可靠、快速简便的遗传检测方法。方法　利用聚合酶链反应 (PCR)扩增技术对 2 0只恒河猴群个体间进行了DNA多态性的分析。结果　筛选出 9个微卫星DNA位点具有显著多态性 ,4个微卫星DNA位点没有多态性 ,还有 2个位点等位基因数目较少。结论　利用这些多态性微卫星位点建立一种对恒河猴群个体进行有效、准备可靠、快捷简便的遗传背景监测方法。     

Directional freezing as an alternative method for cryopreserving rhesus macaque (Macaca mulatta) sperm

The objective was to develop a freezing protocol using a directional freezing (DF) technique for cryopreservation of rhesus macaque sperm and achieve a survival rate comparable to that achieved with a conventional freezing (CF) technique. Rhesus macaque sperm frozen with a DF technique, with cooling rates of 12 or 16 °C/min, had higher post-thaw motility (P < 0.05) than those cooled at 7 °C/min (59.3, 61.1, and 50.3%, respectively). Furthermore, sperm cryopreserved with 5% glycerol and a DF technique had similar frozen-thawed sperm motility to those cryopreserved by a CF technique (63.7 vs. 53.9%, P > 0.05). The function of sperm cryopreserved at the optimized cooling rate using a DF technique was evaluated by in vitro fertilization of oocytes collected from gonadotropin-stimulated rhesus macaques. Of the 38 mature oocytes collected, 78.9% were fertilized and 71.1, 47.4, and 42.1% of the oocytes developed to the 2-cell, morulae, and blastocyst stages, respectively. In conclusion, rhesus macaque sperm was effectively cryopreserved using a DF technique, providing a new and effective method for genetic preservation in this important species.