A simple technique for nonsurgical embryo transfer in mice

A simple method was developed for nonsurgical transfer of mouse embryos which enabled transfer to both uterine horns. Embryos were picked up in a modified capillary tube and transferred through the cervix into each uterine horn of an unanesthetized mouse. A glass speculum was used to facilitate location of the cervix. The technique was found to be as successful (up to 60% of embryos transferred developed to term) as surgical methods yet was simpler and eliminated surgical trauma to the recipient mouse.     

Effect of the bovine viral diarrhea (BVD) virus on preimplantation bovine embryos: A preliminary study

Five crossbred-Holstein cows, approximately three to seven years of age, were superovulated using pregnant mare's serum gonadotropin (PMSG) and prostaglandin F_2α (Prostin F_2α). At the induced estrus, each cow was artificially inseminated with frozen semen. Seven days after insemination, the lumen of the right uterine horn of each cow was inoculated with BVD virus in Eagle's minimum essential tissue culture medium, and the lumen of the left horn was infused with tissue culture medium only. Three days later, each cow was subjected to midventral laparotomy under general anesthesia and embryos were collected. A total of 22 embryos were recovered; 12 were from infected uterine horns and ten were from non-infected uterine horns. All embryos from the non-infected uterine horns were in the late blastocyst stage without the zona pellucida. Of the embryos collected from the infected uterine horns, eight of 12 (66.6%) still possessed zona pellucida and appeared in a degenerative state. The remaining four embryos were morphologically similar to those from the non-infected uterine horns. Electron microscopic examination of the degenerated embryos from the infected uterine horns demonstrated the presence of a structure which morphologically resembled the BVD virus. The results of this preliminary study indicate that the BVD virus within the uterine horns may interfere with normal development of preimplantation bovine embryos. Therefore, it is proposed that the BVD virus could adversely affect early stages of gestation in the cow, resulting in infertility.     

Decidualization in the post-partum uterus of the mouse

Unilaterally ovariectomized mice were allowed to complete one pregnancy before the second ovary was removed and sensitivity to decidualization was induced by hormone administration. The virgin uterine horn and the post-partum horn were stimulated to decidualize by the intraluminal injection of arachis oil. A greater decidual response was found in post-partum horns than in virgin horns. The foci of decidual induction in post-partum horns were regularly spaced reflecting the regular spacing of used and unused areas of the uterus. A focus of decidual induction occurred in 68% of the recently used uterine areas observed, i.e. areas associated with post-partum nodules. When compared with foci of decidualization in adjacent unused areas of the uterus, none of the decidualized used zones showed more decidual tissue, about half showed less decidual tissue and half showed the same amount of decidual tissue. The remaining 32% of used zones were not associated with foci of decidual induction. The results indicate that decidualization can be induced in recently used zones of the uterus using a non-traumatic method of induction, but that used zones are associated with less decidual tissue than unused zones of the post-partum uterus.     

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy

Conceptus-derived paracrine signals play crucial roles in the preparation of a uterine environment capable of supporting implantation and development of the conceptus. However, little is known about the regulation of endometrial tryptophanyl tRNA synthetase (WARS) and manganese superoxide dismutase (SOD2) protein expression by the implanting and post-implanting conceptus. We hypothesized that the conceptus-derived signals favourably influences uterine environment for implantation through regulation of WARS and SOD2 expression in ovine caruncular endometrium. To test this hypothesis, WARS and SOD2 protein and mRNA expression was determined in caruncular endometrial tissues of unilaterally pregnant ewes at implantation (day 16) and post-implantation (day 20) periods. WARS protein expression increased in caruncular tissues of the gravid uterine horns compared with the non-gravid uterine horns on days 16 and 20 of pregnancy. There were no changes in SOD2 protein expression between the gravid and non-gravid uterine horns, irrespective of the day of pregnancy. On day 16 of pregnancy, there were no differences in WARS and SOD2 mRNA expression between the gravid and non-gravid uterine horns but expression of both genes was higher in the gravid uterine horns when compared with the non-gravid uterine horns on day 20 of pregnancy. In conclusion, the use of the unilaterally pregnant ewe model provides for the first time firm evidence that the early implantation and post-implanting conceptus-derived signals up-regulate WARS protein expression within the caruncular endometrium. Further studies are necessary to identify these signalling molecules and to understand mechanisms whereby they exert paracrine action within the endometrium.     

Concentrations of spermatozoa in the vagina of heifers after deposition of semen in the uterine horns, uterine body or cervix

In Exp. I, virgin Holstein heifers (N = 18) were induced into oestrus with PGF-2 alpha. Animals which stood to be mounted were paired for insemination approximately 8 h later with 56.1 x 10(6) spermatozoa from a single bull. Semen was deposited in the uterine body of one female. Each matched female was inseminated by deposition of one-half of the inseminate into the right uterine horn and one-half into the left uterine horn approximately 7.0 cm anterior to the internal cervical os. In Exp. II, additional heifers (N = 18) were induced into oestrus and inseminated by deposition into the uterine horns or cervix (2.0 cm anterior to the external cervical os). A 1.0 ml aspirate of vaginal mucus was collected at hourly intervals for 8 h after insemination. Concentration of spermatozoa was determined by haemocytometry. In Exp. I, cumulative percentage spermatozoa recovered in an 8 h collection period were similar (P greater than 0.10) for insemination into the uterine horns (17.9 +/- 2.9%) and uterine body (18.5 +/- 4.5%). In Exp. II, cumulative % sperm recovery from the vagina was greater (P less than 0.10) for cervical deposition (59.1 +/- 14.1%) than for that into the uterine horns (30.9 +/- 7.8%). In Exp. II, the insemination treatment x hour of sample interaction was significant (P less than 0.08). Recovery of spermatozoa from the vagina was greatest (P less than 0.05) within 3 h after cervical insemination (31.4 +/- 9.9% compared to 9.4 +/- 2.5% for uterine horn deposition). Percentage recovery of spermatozoa from the remaining hourly collections were similar (P greater than 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of side of insemination on transuterine transport of spermatozoa in superovulated dairy cattle

Eleven superovulating Friesian lactating cows were inseminated deep into one uterine horn with one unit of frozen semen, containing 2.5 million total spermatozoa, with more than 40% postthaw progressive motility and with 14% morphologically abnormal spermatozoa. Semen was deposited into the right or left uterine horns of alternate cows. There was no difference in the proportions of fertilized recovered ova from ipsilateral horns between right and left inseminations (P>0.05). The fertilization rate in the contralateral horns was higher (P<0.01) for right uterine horn insemination (50%) than for left uterine horn insemination (15.6%).     

Immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels

Cyclic changes in estrogen receptor expression in the uterine tissue of 60 female dogs were evaluated, using an immunohistochemical technique on formalin-fixed paraffin-embedded sections. The expression of estrogen receptors in the uterine horns, body and cervix was quantified by means of an immunohistochemical score. A negative correlation was found between staining scores in the uterine horns and serum progesterone levels. Generally, staining scores in the uterine horns were highest during proestrus, declined during estrus and were lowest during early metestrus. During anestrus high staining scores for estrogen receptors were observed, indicating sensitivity for estrogens in a sexual quiescence stage. Compared with the uterine horns, high staining scores were found in the uterine body and cervix during estrus and metestrus. No positive staining for estrogen receptors was detected in 1 pregnant uterus. Fluctuations in estrogen receptors were more pronounced in endometrial stroma cells than in epithelial cells of the uterine horns. The importance of stromal cells in the sexual cyclicity of the canine uterus should not be underestimated when studying uterine endocrinology and pathology.     

The rat uterine oestrogen receptor in relation to intra-uterine devices and the oestrous cycle [proceedings

There are changes in the nuclear content of the estrogen receptor in the rat uterus during the estrous cycle that are associated with changes in its physiology. The changes correlate with the concentrations of circulating estradiol. It appears that uterotrophic response to estradiol is a function of the nuclear receptor. The insertion of an IUD leads to changes in the treated uterine horn which appear to be the result of an increased responsitivity to circulating estradiol. The presence of an IUD did not alter the estrous cycle, gonadotropin, or corpus luteum function. The intracellular distribution of the estrogen receptor was investigated in normal uterine horns and in the horns with devices throughout the estrous cycle. Groups of 30 Wistar rats had a silk suture fitted in the lumen of 1 uterine horn. After 14 days the progress of these estrous cycles was determined. Rats were grouped according to the stage of the cycle on the 4th day. Rats were then killed and the uteri removed. Cytosol receptors were measured. The capacity of the cytosol estrogen receptor to bind to oligo(dT)-cellulose was determined. Cytosol protein, nuclear protein, and DNA were measured. At all stages of the estrous cycle, the wet weight and cytosol receptor of the treated horns were greater than the control horns. A slight increase in the capacity of cytosol receptor to bind to oligo(dT)-cellulose was noted at proestrus. The response elicited by the IUD was not considered to be due to an estrogenic response since the changes observed were not accompanied by a corresponding increase in the content of nuclear receptor.     

Growth and microvascular development of the uterus during early pregnancy in ewes.

Growth and microvascular development of the uterus were evaluated for ewes on Days 12, 18, 24, and 30 after mating (3-4 ewes/day; Day 0 = day of mating) in two experiments. In experiment 1, fresh weight and dry weight of gravid uterine horns were increased on Days 24 and 30 after mating, whereas those of nongravid uterine horns were elevated only on Day 30. The increased fresh and dry weights of gravid uterine horns on Day 24 were associated with uterine hyperplasia (increased DNA content). Increased fresh and dry weights of gravid uterine horns on Day 30, however, were associated with hypertrophy (increased RNA:DNA and protein:DNA ratios) of uterine tissues. In experiment 2, vascularity of endometrial tissues was elevated on Days 24 and 30 after mating. In addition, dramatic changes in uterine architecture (increased lumenal diameter and decreased endometrial thickness) and in uterine microvascular development (increased abundance of large microvessels and development of a subepithelial capillary plexus) were observed by Day 24 after mating. Characterization of the patterns of uterine growth and microvascular development will enable us to further define the role of previously reported uterine and conceptus-derived growth and angiogenic factors during early pregnancy.     

Inequality in function of the right and left ovaries and uterine horns of the mouse

Inequality in function of the left and right ovaries and uterine horns of mice was evaluated in three separate experiments. In Exp. 1, the effect of position in the reproductive tract on various reproductive characteristics was evaluated in 158 pregnant hybrid mice. Ovulation rate, number of fetuses, total fetal weight and total placental weight were higher (P less than 0.05) on the right than the left on Day 18 of pregnancy (vaginal plug = Day 1). In Exp. 2, the effect of previous sham or unilateral ovariectomy (right or left) in mated Swiss-Webster mice was compared with unoperated mated controls (N = 17-24/treatment). In control mice, ovulation rate, total fetal weight and ovarian weight were higher (P less than 0.05) on the right than left side. Surgery (sham or unilateral, ovariectomy) decreased (P less than 0.005) ovulation rates, number of fetuses, ovarian weights, total fetal weight and total placental weight on Day 18 of pregnancy. Unilateral ovariectomy decreased (P less than 0.05) ovulation rates and ovarian weights more than did sham operation. Ovulation rates were higher (P less than 0.01) when the left ovary was manipulated or removed rather than the right ovary. For Exp. 3, pairs of 8 hybrid mouse embryos each (morulae and blastocysts) were surgically transferred to the left and right uterine horns of the same (bilateral, N = 15) or different (unilateral, N = 28) Swiss-Webster recipients. In almost all incidences, embryo survival (to Day 18 of pregnancy) was twice as high (P less than 0.05) in right than left uterine horns. We conclude that the left and right ovaries and uterine horns are not equal in function in Swiss-Webster and a hybrid strain of mice.     

Effects of unilateral ovariectomization on the numbers of sperm in the oviducts, uterus and cervix of rabbits

The experiment was designed to determine the effect of removing one of the rabbits' ovaries on the numbers of sperm in the oviducts, uterine horns and cervices. The possible relationship between the anatomical asymmetry of the two sides of the reproductive tract and the distribution of sperm within different segments of the tract was also studied. Fifteen female rabbits were used; five were kept intact as the control group, five were right ovariectomized and five were left ovariectomized. All rabbits were injected with 50 IU HCG to induce ovulation and then inseminated with 60x10(6) sperm in 0.25 ml semen. Does were inseminated one month after unilateral ovariectomy. Animals were sacrificed 10 hrs later and sperm was recovered from the right and left oviducts, uterine horns and cervices. Unilateral ovariectomy significantly reduced the total numbers of sperm recovered as compared with intact does. The total numbers of sperm recovered from each of the two sides of the tract were not affected by the site of the removed ovary. Sperm numbers were high in the cervices of all groups and then decreased gradually in the upper segments of the tract. Sperm numbers that reached the oviduct of the intact rabbits were greater than those of both unilateral ovariectomized groups of rabbits. Differences between the length of the left and right cervices and uterine horns were not significant, but the right oviduct was significantly longer than the left oviduct.     

Is the spontaneous motility of isolated rat uterus controlled by prostaglandin E?

Variations in the spontaneous contractile activity during 6 hours following isolation of uterine horns from proestrus, metestrus and spayed rats, were explored. In estrus and metestrus preparations the contractions declined during 60 min and between 180--200 min a progressive spontaneous recovery (abolished by indomethacin) was observed up to 360 min. Uteri from proestrus and spayed animals exhibited a continuous depression without recovery during the whole experimental period. At 60 min, uterine horns from estrus animals (which showed a marked contractile decrement) released to the suspending medium significantly less prostaglandin E-like material than at 360 min, i.e. when contractions had almost completely recovered. No modification in the amount of prostaglandin F-like material was detected accompanying these spontaneous contractile variations. In the spayed group at 60 min of functioning (i.e. when the contractile impairment was significantly smaller than at a later time) the release of PGE was greater than at 360 min. These findings suggest a possible control of rat uterine contractions by PGE, rather than by PGF.     

Equine fetal kinetics: Allantoic-fluid shifts and uterine-horn closures

Uterine filling by the conceptus and uterine constrictions and allantoic-fluid shifts after filling were studied by daily ultrasound examinations on Days 40 to 74 in 10 mares. Allantoic fluid was first detected at the extremities of the uterus on the following days: 1) tip of umbilical-cord horn, Day 60.0 +/-1.9 (mean +/-SEM); 2) tip of noncord horn, Day 64.8 +/-1.3; and 3) near cervix, Day 48.5 +/-1.3. Transient uterine constrictions with expulsion of allantoic fluid were detected in 37% of 271 examinations. Other end points were studied by weekly ultrasound examinations from Day 40 to term. Height of cross-sections of the fetal body increased (P<0.01) progressively over Monthes 2 to 8, whereas the height of placental fluid minus fetal body decreased (P<0.05) after Month 3. An increasing (P<0.05) frequency of examinations with a reclosed uterine horn occurred between Months 3 and 4. The frequency of reclosed noncord horns continued to increase (P<0.05) after Month 4 and reached 96% at Month 7. The frequency of reclosure of cord horns plateaued (no significant differences) at 54% over Months 4 to 7 and then rapidly decreased (P<0.05). Both uterine horns were closed during the examination that preceded the permanent entry of the fetal hind limbs into the cord horn in all of 9 mares in which this temporal relationship was studied. After entry of the fetal hind limbs, the cord horn remained constricted upon the limbs until at least Month 10. The results provide rationale for the following hypotheses: 1) The gradual decrease in fetal mobility (location and presentation changes) previously reported for Months 4 to 8 is associated with growth of the fetus and a concomitant decrease in volume of allantoic fluid and number of open uterine segments; 2) Closure of both uterine horns near mid-pregnancy plays a role in final selection of cranial fetal presentation followed by entry of the fetal hind limbs into the cord horn.     

Effect of progesterone on the spontaneous motility and prostaglandin synthesis in uterine horns isolated from ovariectomized rats

The motility of isolated uterine horns as well as the generation of PGE and PGF-like material by uterus from spayed rats, treated or untreated with progesterone or progesterone plus estradiol-17-beta, were studied. The changes of Functional Activity (FA) with time (constancy) of control uterine horns and that preparations treated wtih 2 mg of progesterone (P) were not significantly different. However, the PGF-like material released into the bathing solution was significantly higher when the animals were treated with P. PGE-like material in the medium was similar in both groups. With higher doses to P (4 mg/day/2 days) the constancy of FA was similar to that observed in untreated animals, and the PGF-like material released into the medium was significantly higher than in the control group FA and PGs releases into the bathing medium by uterine horns from supra-renalectomized-ovariectomized animals (treated or not with P) were similar to those obtained in spayed rats with the intact suprarenal gland, but the absolute values of PGF-like material were always lower than in this group. Estradiol-17-beta injected prior or after P diminished the stimulation induced by P on the release of PGF-like material into the medium. The constancy of the contractile activity as well as the uterine release of PGE-like material was also diminished in rats treated with P plus estradiol-17-beta. The novel finding that progesterone stimulates the synthesis of PGF in uterine horns from ovariectomized rats without changing that of PGE is discussed.     

The roles of progestagen and uterine irritant in the maintenance of cystic endometrial hyperplasia in the canine uterus

Cystic endometrial hyperplasia (CEH) was induced in the left uterine horns of 14 mature ovariectomised greyhound bitches with an intra-luminal silk suture (uterine irritant) and treatment with estradiol benzoate and megestrol acetate (to simulate stages of a normal canine estrous cycle). Right uterine horns served as suture-free controls. From Day 30 of simulated diestrus, bitches received treatments of suture removal (n = 4), progestagen withdrawal (n = 5) or both (n = 5). Necropsies were performed 3 or 9 weeks later. At 3 weeks, severe cystic endometrial hyperplasia was present in all (6/6) left horns and in no (0/6) right horns. At 9 weeks, the left horns in 5/6 of bitches subjected to progestagen-withdrawal had recovered (in varying degrees) from cystic endometrial hyperplasia, whereas no recovery was evident in the left horns of bitches (n = 2) that continued to receive progestagen. This study demonstrated that: (i) cystic endometrial hyperplasia was reversible upon withdrawal of progestagen; (ii) progestagen maintained cystic endometrial hyperplasia in the presence or absence of irritant; and (iii) persistent endometrial irritation in the absence of progestagen may not maintain cystic endometrial hyperplasia.     

An improved procedure for the preparation of rat uterine cell suspensions.

In the present paper we report on an improved procedure for the preparation of free uterine cells which avoids the use of trypsin and employs very low concentration of collagenase. The cells released mechanically from the digested tissue are constantly removed from the enzyme containing medium, thus minimizing exposure to collagenase. 60%-70% of the cells which make up the intact uterus are obtained as free cells and 95% of these cells are viable for at least 15 hours at 37 degrees. Metabolic integrity was assessed by measuring the cell's ability to oxidize glucose and synthesize proteins over extended periods of time. The membrane leucine carrier protein and the membrane Na+/K+ ATPase were found to be fully functional. Electron microscopic analysis of the cells confirmed their structural integrity. Data are presented illustrating that with this system the estrogen binding protein is stable at physiological temperatures. The cells contain approximately 30,000 specific estrogen binding sites, with an apparent KA of 5--6 x 10(9) M-1. At 37 degrees 80% of the hormone receptor complexes were in the nuclear fraction, 20% in the cytoplasm. The similarity of the estrogen receptor binding parameters with those measured in the intact tissue after in vivo hormone adminsistration, together with the cells' structural and metabolic integrity make this procedure for the preparation of uterine cell suspensions in high yields particularly suitable for studies in which minimal cell injury is an essential prerequisite.     

Biological and synthetic prostheses in gynecologic microsurgery]

Biological prostheses of human umbilical vein and synthetic prostheses of ePTFE were comparatively studied in the microsurgical anastomosis of the uterine horns in twenty rats. Patency rate after 30 days was 60% for umbilical vein and 75% for the ePTFE prostheses. Gross and histological examination of umbilical vein graft specimens showed minimal alteration with a smooth intimal surface. On the contrary tissue covering the inner surface from the adjacent ends of the uterine horns was noted in ePTFE prostheses.     

Effects of intrauterine foreign body on the localization of ( 3 H)-estradiol in rat uterine tissues, polymorphonuclear neutrophiles and eosinophiles

The effect of an intrauterine silk suture on localization of tritiated estradiol in the rat uterus was observed by dry autoradiography. 7 2.5 month old rats were ovariectomized and fitted with a 5-0 silk suture in one uterine horn. After 2 weeks, they were injected with 1 mcg estradiol 17-beta sc daily for 4 days, and 2 days later with .1 mcg per 100 gm body weight of 2,4,6,7-tritiated-estradiol-17-beta (specific activity 95 Ci per mM. Uteri from 6 rats were frozen-sectioned and mounted on dry emulsion-coated slides, at 10 and 60 minutes after sc injection, and 2 minutes after an iv injection in 1 rat. Autoradiograms observed 157 days later showed silver grains concentrated over nuclei in the outer layers of lumenal and glandular epithelium, substantia propria and muscularis in control horns. In uterine horns containing sutures, lesser radioactivity was observed in the basal epithelium and substantia propria, and higher activity in the nuclei of the stromal and glandular cells. No uptake was apparent in the eosinophiles or polymorphonuclear neutrophils which accumulated in the intrauterine suture horns.     

Transplacental diffusion in a bicornuate uterus: comparison of uterine blood flow and oxygen uptake between horns

The purpose of this study was to determine whether samples from the veins of the pregnant and the nonpregnant horn of the uterus lead to similar estimates of uterine blood flow and oxygen consumption. To accomplish this, a comparison of uterine blood flow, arteriovenous differences of oxygen content, and oxygen consumption measured by sampling the venous drainages of the two uterine horns was performed on eight pregnant sheep during the last 20 days of pregnancy. Each sheep carried a single fetus. Umbilical and uterine blood flows were measured with the test substances ethanol and antipyrine by application of the steady-state diffusion method. Twenty-three measurements of uterine blood flow comparing the two horns were not significantly different (P greater than 0.1), and were highly correlated (r = 0.98). The ratio of the oxygen content arteriovenous difference in the pregnant to that in the nonpregnant horn and the ratio of the uterine blood flow in the nonpregnant to that in the pregnant horn were significantly correlated (r = 0.7). As a consequence, paired calculations of oxygen consumption for the whole pregnant uterus had a small coefficient of variation (+/- 3.7%). These results demonstrate that the use of highly diffusible test substances for the measurement of uterine blood flow in pregnant sheep can provide accurate data for the calculation of uterine oxygen uptake, in part because the oxygen and test substance molecules are similarly affected by local variations in placental perfusion.     

Distension of the uterus induces HspB1 expression in rat uterine smooth muscle

The uterine musculature, or myometrium, demonstrates tremendous plasticity during pregnancy under the influences of the endocrine environment and mechanical stresses. Expression of the small stress protein heat shock protein B1 (HspB1) has been reported to increase dramatically during late pregnancy, a period marked by myometrial hypertrophy caused by fetal growth-induced uterine distension. Thus, using unilaterally pregnant rat models and ovariectomized nonpregnant rats with uteri containing laminaria tents to induce uterine distension, we examined the effect of uterine distension on myometrial HspB1 expression. In unilaterally pregnant rats, HspB1 mRNA and Ser(15)-phosphorylated HspB1 (pSer(15) HspB1) protein expression were significantly elevated in distended gravid uterine horns at days 19 and 23 (labor) of gestation compared with nongravid horns. Similarly, pSer(15) HspB1 protein in situ was only readily detectable in the distended horns compared with the nongravid horns at days 19 and 23; however, pSer(15) HspB1 was primarily detectable in situ at day 19 in membrane-associated regions, while it had primarily a cytoplasmic localization in myometrial cells at day 23. HspB1 mRNA and pSer(15) HspB1 protein expression were also markedly increased in ovariectomized nonpregnant rat myometrium distended for 24 h with laminaria tents compared with empty horns. Therefore, uterine distension plays a major role in the stimulation of myometrial HspB1 expression, and increased expression of this small stress protein could be a mechanoadaptive response to the increasing uterine distension that occurs during pregnancy.