A microscopical study of uterine lining modi®cation, binucleate cell
numbers and trophoblastic development, at day 14, 20 and 24 of
gestation in single and multiple pregnancies in sheep
I. RodrõÂguez
a, C. JimeÂnez
b, A. HernaÂndez
b,*aDepartment of Animal Biology, School of Veterinary Sciences, University of Zulia, Maracaibo, Venezuela
bDepartment of Physiological Sciences, School of Veterinary Medicine and Animal Production, National University, BogotaÂ, Colombia
Accepted 24 May 1999
Abstract
In order to evaluate possible differences in some key anatomical characteristics of the placental components between single and multiple pregnancies in sheep, mean percentage values of trophoblastic development (TD), binucleate cell numbers (BC) and modi®cation of the uterine lining epithelium (ULM) were obtained in ewes carrying one (natural single pregnancies; Group 1) and two or more embryos (multiple pregnancies obtained by superovulation; Group 2) at 14, 20 and 24 days postmating. Two sheeps per stage were used in Group 1, and three in Group 2 were employed. TD, BC and ULM mean values were higher at all stages in Group 2. Under the conditions of the present study, it appears that in multiple pregnancies, implantation ensues earlier compared to in singles, and the correspondent anatomical changes in the conceptus and endometrium are more advanced in multiple gestations. A possible explanation for the present ®ndings is that each embryo locally in¯uences the development of the above mentioned modi®cations at day 20 and 24 postmating. In multiple pregnancies at day 14, it appears that the degree of development differs among conceptuses con®ned to the same uterine environment. The present studies corroborate previous ®ndings that a critical phase during implantation for embryonic survival might be the time period between 14 and 24 days of pregnancy due to the gradual and relatively slow nature of the implantation phenomenon. It can be stated that TD, ULM and BC are important histological features to evaluate embryonic viability in postmortem assessments.#2000 Published by Elsevier Science B.V. All rights reserved.
Keywords:Trophoblast; Uterus; Sheep; Implantation; Superovulation
1. Introduction
Most embryonic losses occur in early pregnancy (Edey, 1969), which covers the time period from fertilization to about day 30 of gestation (Zheng et al., 1998). The understanding of the physiological
and morphological events taking place during im-plantation might become salient cues to know how embryo survival or deaths occur and could then permit the development of procedures that might enhance pregnancy rates.
It has been established that implantation in the ewe is a gradual and long lasting process, ensuing near the embryo, and spreading over to more distant zones of the conceptus as pregnancy progresses (HernaÂndez, *Corresponding author. Tel.: +57-3165-402; fax: +57-3165-044.
E-mail address:[email protected] (A. HernaÇndez).
1971; Gaviria and HernaÂndez, 1994; Guillomot, 1995). The number of binucleate cells (BCs) is given as a criterion to identify viable conceptuses, although values are not given at a de®nite gestational stage (Boshier, 1968). Furthermore, it has not been reported when the BC appear in the trophoblast and whether or not they are subjected to numerical variations as pregnancy progresses.
The development of the trophoblast (change to a cuboidal or columnar two layered epithelium from a simple cuboidal layer) and the modi®cation of the uterine lining epithelium (from a columnar to a ¯at-tened tissue) were found to be typical histological changes of successful implantation. These modi®ca-tions occur between days 14 and 24 of gestation (HernaÂndez, 1971; Gaviria and HernaÂndez, 1994). In agreement to this, the ®rst histological changes of implantation in sheep were reported to ensue at day 15 of pregnancy (Guillomot, 1995).
Most of the information regarding implantation has been performed in pregnant ewes carrying singletons, but there is little or no information about morpholo-gical dynamics of early implantation in multiple pregnancies. This is of special interest because intrau-terine competition among conceptuses could change trophoblastic development and affect embryo or fetal survival, although critical periods in this context have not been established.
The present study was designed to examine the degree of trophoblast development (TD), the number of binucleate cells (BCs), and the modi®cation of uterine lining epithelium (ULM) during implantation in single and multiple (superovulated) pregnancies in sheep.
2. Materials and methods
The work was carried out in BogotaÂ, Colombia (altitude: 2638 m above sea level; average annual temperature: 138C; relative humidity: 79%; rainfall: 938 mm per year). Fifteen sexually matured crossbred ewes were used in this study, fed with natural pasture (Pennisetum clandestinum), using a daily supplement of 250 g per animal of concentrated commercial feed containing 14% protein and mineralized salt and water. The ewes were randomly allocated in three different stages of pregnancy: day 14, 20 and 24 of
gestation (day 0estrous detection). Within each group, the ewes were allowed to have a single preg-nancy (NSO) or were superovulated (SO). The NSO group had two animals per stage, whereas three ewes (per stage) were included in the SO group. In the SO group, each animal had at least a total count of ®ve corpora lutea. Animals with less CL counts were discarded.
Sheep in the NSO group were allowed to naturally mate after two naturally occurring estrous cycles. All animals in the SO group, were synchronized for estrus (after a third naturally observed estrus), using intra-vaginal sponges, containing 60 mg of medroxypro-gesterone acetate,1for 13 days. The SO group received 1500 IU (im) of PMSG,248 h prior to sponge with-drawal. Sheep in this group were also naturally mated. Estrous detection was assessed by direct observation with the help of a caudo-epidectomized ram.
Slaughter was carried out by exsanguination after desensibilization. The genital tracts were immediately removed after death. Fixation of the organs was carried out by perfusion through the middle uterine and vaginal arteries with a 3% gluteraldehyde solution (pH 7.4). Each uterine horn was divided into three equidistant segments in addition to one taken from the uterine body. Transversal samples were obtained at each division point for tissue processing and paraf®n imbibition procedures after post®xation with a 10% buffered formalin solution. 5mm sections in diameter (7 sections per ewe) were stained with Hematoxylin and Eosin (H&E) and PAS and hematoxylin standard procedures (Luna, 1968).
The following calculations were made on each histological preparation:
1. TD calculations. A developed trophoblast was taken as an epithelium with at least two layers of cuboidal and/or columnar cells. An epithelium of the single cuboidal type was considered to be a non-developed trophoblast (HernaÂndez, 1971; Gaviria and HernaÂndez, 1994).
2. The number of binucleate cells (BCs) (using the PAS and hematoxylin stained sections) were expressed as a percentage of the total number of cells counted in 10 randomly chosen microscopical
fields. For this purpose, a light microscope was employed using a 100X objective.
3. ULM was expressed as a percentage of flattened epithelia in a given area. The total area of the lining epithelium of the uterus in each histological pre-paration was studied using the direct option of a computerized image analyzer.3 Mean values for each animal were calculated from calculations obtained from the total available area on each one of the seven sections analyzed per uterus.
Descriptive statistics were brought about using the statistical analysis system (Statistical Analysis Sys-tem, 1985).
3. Results
At 14 days of gestation, TD, BC and ULM values were 0 in tissues from the NSO sheep (Table 1). In the
uteri containing several embryos at day 20 and 24 (mean5.5 embryos/uterus), two of the three ewes showed some degree of TD and ULM. Nevertheless, BC values were 0 (Table 2).
At day 20, both single and multiple pregnancies clearly showed increments in the values of TD, BC and ULM in comparison to the observations made at 14 days of pregnancy. The three SO ewes showed greater values of TD, BC and ULM than the NSO treated ewes, although there was great variation in the obtained results (Tables 1 and 2).
At 24 days of gestation TD and ULM were not complete (i.e. less than 100%) all over the potential placental area in single and multiple pregnancies, but an increment with regard to observations at 20 days could be seen. Both groups of animals showed greater modi®cation when compared to the 20 day groups. BC increased from 20 to 24 days of pregnancy and were higher in the SO group. Changes in BC and ULM were more marked in the SO compared to the NSO treated ewes regarding the TD values (Tables 1 and 2). TD mostly occurred between 14 3Leco 2001, Leco, USA.
Table 1
Percentage values of TD, BC and ULM in single pregnancies at days 14, 20 and 24 in sheep (meanSE)
Animal number Day of gestation TDa BCa ULMa
2016 14 0 0 0
10114 14 0 0 0
12114 20 70.617.6 9.06.7 34.334.2
3614 20 52.339.6 7.24.2 24.341.0
2438 24 74.925.4 9.32.5 44.943.5
2322 24 75.335.8 8.23.5 54.341.3
aTD: trophoblastic development. BC: binucleate cell numbers. ULM: modification of the uterine lining epithelium. (X SE): meanstandard error.
Table 2
Percentage values of TD, BC and ULM in multiple pregnancies at days 14, 20 and 24 in sheep (meanSE)
Animal number Day of gestation TDa BCa ULMa
2318 14 4.13.6 0 0.852.1
2436 14 4.43.0 0 0.671.2
2638 14 0.81.8 0 0.140.3
1004 20 73.06.7 11.13.7 67.539.4
2114 20 82.512.0 12.23.4 77.837.6
4114 20 80.019.4 12.26.0 75.638.3
4404 24 77.415.4 20.49.8 84.431.3
22113 24 78.36.3 16.67.2 83.332.8
20493 24 92.38.3 17.75.8 96.36.5
and 20 days of gestation, showing little variation between day 20 and 24. At 14 days of gestation the trophoblast consists of a single layered epithelium which becomes a predominantly double layered tissue during the 14±20 days of the gestation period, although it had not reached full development at day 24. TD and ULM in SO ewes appear to start earlier in gestation than in NSO ones since there was some degree of modi®cation at 14 days of gestation, which was not seen in the single pregnancy group (Tables 1 and 2).
4. Discussion
The observed differences in TD and ULM at day 14 of gestation between single and multiple pregnancies could be interpreted as the presence of conceptuses in a more advanced stage of development in the uteri of superovulated sheep as a consequence of the presence of more than one embryo. It is possible that embryos could respond to intrauterine competition by accel-erating the implantation process in order to assure their nourishment and survival. TD and ULM values at day 24, in single and multiple pregnancies, were lower than those previously reported for the same stage (Gaviria and HernaÂndez, 1994). This emphasizes the irregular character of implantation, probably re¯ecting genetic differences in survival potential, and/or the presence of conceptuses in the process of degenerating.
These ®ndings as a whole could also imply that some embryos have a more rapid rate of development as seen in earlier stages in mice, where a speci®c gene was identi®ed, which causes enhanced mitotic rates in blastomeres (Brownell and Warner, 1988). In sheep and cattle, it is a common ®nding to encounter quite different sizes of the conceptuses at the same age (Rowson and Moor, 1966; Gaviria and HernaÂndez, 1994; Escobar and HernaÂndez, 1996).
Several works point out that trophoblastic development could be controlled by the secretion of IGF I and II, and EGF acting in autocrine or paracrine manners (Geisert et al., 1991; Simmen et al., 1993; Gharib-Hamrouche et al., 1993). Differences in the effects of these growth factors, due to differences in physiological characteristics derived from speci®c genetic expressions, could explain the observed
var-iations. In this context, it has been demonstrated that mitogens are present in the endometrium of ewes during early pregnancy controlling vasculogenesis (Reynolds and Redmer, 1992; Zheng et al., 1998), which are liable to participate in the process of trophoblastic development and BC formation. It is possible that those molecules together with other growth factors are present in greater amounts in multi-ple pregnancies, thus accelerating TD, and conse-quently ULM. At 24 days of gestation, histological changes of implantation are clearly identi®able in the majority of the area analyzed in the present study, which implies the presence of a well-developed tro-phoblast and more than 90% of the uterine lining epithelium already modi®ed to a ¯attened tissue by the presence of the trophoblast. Appearance of BC, trophoblastic growth and attenuation of the uterine lining epithelium are all events taking place during this period of time. Therefore, it seems that implanta-tion can be more easily disrupted between 15 and 24 days of gestation than at more advanced stages of pregnancy.
Binucleate cells have been considered as an indi-cator of embryonic viability during implantation (Boshier, 1968). The present ®ndings support the central role of these cells in the implantation process claimed by several authors (Wooding, 1984; Guillo-mot, 1995; Wooding et al., 1996). However, it appears that binucleate cells are not necessary to initiate implantation, in the multiple pregnancies studied, given that at 14 days of gestation, proper changes of implantation have ensued in spite of the absence of BC. Nevertheless, since BC could move into the endometrial wall, it is also possible that all BC were not identi®able in the analyzed sections and/or were present in non-studied areas.
trophoblast is the histological picture to ®nd in viable conceptuses.
Differences in ULM and TD at 14 days of gestation could imply that implantation tends to be more advanced in viable conceptuses subjected to intrau-terine competition in virtue of the high number of conceptuses. Perhaps this is a mechanism against adverse situations. In fact, evidence has been given that implantation is dependent on the activity of BC which are entities secreting fundamental molecules to embryonic survival (Wooding, 1984; Wooding et al., 1996).
It could be inferred that the employment of chor-ionic gonadotropin for superovulating the animals employed in the present study might interfere the microenvironment of a developing embryo and thus with implantation. This hormone has been shown to participate in implantation because there are hCG receptors in the uterus (Rao and San®lippo, 1997). It should be pointed out that hCG was administered with suf®cient time ahead of the time of sheep slaugh-ter so as to allow for its metabolization. It cannot be said with full certainty that a conceptus of doubtful viability was present in the uteri of superovulated sheep. Nevertheless, histological changes of embryo-nic decay were not found in the present work accord-ing to the light histological characteristics proposed by Boshier (1968) (atrophic trophoblast, absence of BC) and HernaÂndez (1971) (absence of modi®cation of the uterine lining epithelium).
Although some interesting anatomical characteris-tics and correspondent variations are presently reported, in regard to key aspects of successful or failing implantation, many questions remain unan-swered relating to the control of implantation. Speci-®cally, the interaction or interdependence of genetic (embryonic and/or maternal) and secretory factors mastering this biological phenomenon.
Under the conditions of the present study, it appears that in multiple pregnancies, implantation ensues ear-lier than in single ones. The correspondent anatomical changes in the conceptus and endometrium are also more advanced in multiple gestations. These ®ndings could be due to individual differences among embryos and/or speci®c local in¯uences of an embryo on the uterine lining epithelium, which in turn, would sum up in multiple pregnancies so as to mimic more advanced stages of implantation.
5. Conclusions
The present studies corroborate previous ®ndings that a critical period during implantation for embryo-nic survival encompasses the time period between 14 and 24 days of pregnancy. It can be stated that TD, ULM and BC are important histological features to evaluate embryonic viability in postmortem assess-ments.
Acknowledgements
The authors are deeply indebted to Dr. Amparo Cortes for professional assistance. This research was ®nancially supported by the National University of Colombia, Colciencias and the University of Zulia, Venezuela.
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