Early embryonic development in sheep and goats

G.M. Wani, Khatoon A., P.Gowsomi, S. Mir, Darzi M.M and M.Z.Khan
S.K.University of Agricultural Sciences and Technology(K)
Directorate of Extension Education Shalimar

Introduction

Sheep uteri consist of a large number of caruncles. These caruncular areas are the sites of implantation. (Gray et al, 2002). Ovine preimplantation conceptus undergoes elongation. The elongated blastocyst extends itself both in gravid and non-gravid uteri. Uterine epithelium under goes through different changes prior to implantation. What is the nature of these changes and how they can be morphologically recognized is still obscure. The placentomes serve as immunological barrier membrane and mediate gas and micro-nutrient exchange. The changes under the influence of corpora haemorrhagicum is the beginging of immunological protective cover, which prevents rejection of the cenceptus as a foreign body or influences maternal recognition of pregnancy (Wani, 1996).

This early development mechanism is our primary concern to be investigated in this study. Perhaps this invisible mechanism was populated by Corner back in 1947. He said, "Anterior chamber of eye is superior to womb for nourishing early embryo except that the ovarian hormones make it a well cherished superior chamber for survival and growth of the embryo."

Attempts to describe this superiority of "Womb" have remained limited to endocrinological and hormonal explanations and explorations. This investigation attempts to unravel the morphological and cytological aspects to explain the mechanism of protection, placenta development, immunological barrier and implantation signals in ovine conceptus.

Paper presented on 2nd J&K Science Congress, held at Kashmir University in July,2006
Objectives

The embryonic diapauses model of delayed implantation in lactating mothers was thought to give important clue on the subject. (Lopes et al 2004).. Morphological and cytological studies with endocrinological and other physiological aspects explanations is not available to find basic answer to many questions like:

1. Role of corpus luteum in initiating caruncle and cotyledon development.
2. Syncytia formation and blastocyst elongation.
3. Binucleate and hybrid nucleate cell formation. As such this study has investigated.

a) The local uterine vascular permeability and receptivity of uterine epithelium and its morphological and cellular visualization. It was the primary focus for this investigation.
b) This study shall gather information on signaling pathways and co-relate it with visual post-mortem and slaughter house samples. All these studies on abattoir materials would be collaborated through in vivo-ultrasonic and electronic images to be posted in literature later, as work is in progress.

Brief Review:

Endometrial receptivity is stimulated by ovarian steroids. The role of ovine and caprine ovarian steroids in initiating endometrial receptivity have been reviewed by various workers.( Huct-Hudson et al, 1989, Wani, 1995, 1996 Wani, 2003 , Wani 2005, Wani, 2006). Proliferation of uterine cells is influenced by E2 and P4. These hormones have their cognate nuclear receptors in ovine/Caprine uteri called E2 receptor (ER) and P4 receptor (PR). {Lee, and De Myo, 2004). This intrinsic ability of uterine epithelium has been reviewed (Dey et al 2004).

The intercaruncular regions filled with uterine glands in sheep develop during Ist month post Lambing. A simple tubular luman is replaced with aglandular, carcuncle inhabited or projected uteri as shown in our prdiminary investigations Fig.1 Wani et al 2006 and others too {Wiley et al, 1987). These uterine glands fail to develop if pre-natal withdrawal of P4 at birth was removed. This mean at birth the P4 supplies to new born ewe lamb hinders uterus glandular formation due to lack of progesterone stimuli. In an overetomized new born ewe lamb, implants of 19 norprogestin, failed to develop uterine glandular system. Thus "Progesterone knock out" at birth stimulates glandular formation during first month of newborn ewe lamb. These experiments on prolonged use of P4 stimulation in neonate ewe lambs has lead to a model commonly called as "Ovine uterine gland Knockout (UGKO)." Animal model. (Spencer et al, 1999). This model has been used to identify gene expression in endometrial epithelium (Gray et al 2002).

Gene expressions under the stimuli from P4 are essential for implantation and maintenance of pregnancy. Various other factors having significance for inducing implantation are calcitonin, Indian hedge Log (IHA) Ambhiregulin etc (Lee and Dehayo, 2004).

Various growth factors like epidermal growth factors and their family members like TGF2 (Transforming Growth factor 2) released during pre-implantation, heparine binding EGF (HB-EGF) related to blastocyst attachment in mice and other factors like vascular endothelial growth factors (VEGF) reviewed by Wani, 2006 and described by Tamada et al 2002, Paria et al 2001, Tischer et al, 1991 need exploration in dertermining ovine implantation mechanism. Various studies in humans (Mueller et al, 2000, Wang et al 2003 Row et al 2003) could help to develop ovine-implantation-mechanism model envisaged in this study. The role of cytokines (Hilton, 1998) like Leukemia inhibitory factors (LIF) and others like interleukins-II; colony stimulating factor-1 (CSF1) and Modulators cell adhesions Mucin I (MucI) are important biochemical parameters to be probed for explaining ovine implantation model.

Developmental factors like Homesbox (Hox) genes, Wnt genes could help to understand regulatory and signaling processes involved in ovine implantation. Many such studies have been undertaken in mice or primates. (Tulac et al 2003) but remain obscure in ovine and human uteri. This implantation mechanism knock out is the primary objective of this study. Once understood embryonic mortality and fertility improvement in livestock shall be easier. It may help to devise side affect free biological control of human fertility. Its economic importance can be counted in millions of dollars and human welfare, thus this study shall have both economic and human welfare prospective.

The following innovative observations accredited to our group are detailed as follows. This innovative lead paper shall describe and show our pioneering and original results. To the best of our knowledge no such observations on foetal development are available in literature.

As we all know sheep and goat fertilized eggs develop into a Morula (8 cells) within 2-3 days post coitus. The figures 1 – 2 shows the oocytes and morula of sheep cultivated in our laboratory (Wani, 1996, Wani et al, 2000}. The corpus luteaum Haemorrhagium (CLH) can be seen in Fig. 3. The early uterine epitheliaum under the influence of P4 secretions from functional corpus luteaum can be seen in Fig. 4. This is a clear angiogenic activity in early pregnancy and indicates preparation of the uterine epithetheluim to accommodate the incoming conceptus. On the other side of the figure marked A is another figures showing functional CL at mark "B". Fig.5 shows the persistent corpusluteum (PCL). The hypothetical attachment site in sheep can be seen in Fig. 6. These are the elongated foetal blastocyst. The foetus lies within amniotic sac. The reddish areas are the implantation site as observed in our investigation. An external view of 25 & 57 day old foetus within maternal uterus can be seen in Fig. 7. The raised nodes on the large sac(A) are developing. The caruncle in a non-pregnant uterus can be seen in Fig. 8.

There is a wide confusion among biologists about the three terms, caruncles, cotyledons and placentomes. Our observations are the first to show a vivid distinction between the three. Caruncle can be seen as solid projections on the surface of the uterine epithelium as shown in Fig. 8. They may be hypertrophied in some disease conditions or during normal pregnancy of early 2 week as shown in Fig. 8(a). The early foetal development in an elongated blastocyst before attachment can be seen in Fig. 9. The histological evidence of the union of foetal placental membranes with the uterine caruncle can be established only when the foetal cotyledons as shown in Fig. 10 get attached with maternal cornucle Fig.8. This results into an intimate association with the mother in the form of placentomes as show in Fig. 11.

The development of the foetus during first two weeks takes place within the foetal membranes as shown in Fig. 12 & 13. The corresponding uterine epithelial changes during first week of gestation can be increased blood supply to the uterus as shown in Fig.14. The corresponding histological evidences of syncytium development in early pregnancy can be seen in Fig. 15 – 20. These findings shall be discussed in the presentation.

References:

Gray, C.A; Barghardt, R.C; Johnson, G.A; Bazer, F.W. and Spencer, T.E 2002. Evidence that absence of endometreal gland secretions in uterine gland Knock-out ewes compromises conceputs survival and elongation Reproduction. 124:289-300

Wani, G.M. 1996. Embryo Biotechnology in Sheep and Goats. Publisher Valley Book House, University Road, Hazratbal Srinagar – Kashmir

Corner, G.W. 1947. The hormones in human reproduction. Ed; Princeton N.J.Princeton University Press.

Lopes, K.Y. and De-Mayo, F.I. 2004. Animal Models of Implantation Reproduction 128:679-695

Huct-Hduson, M.Y.Andres, G.K. and Dey, S.K. 1989. Cell type specific localization of C-Myc-Protein in mouse uterus-modulation by steroid hormones and analysis of the pre-implantation period. Endocrinology. 125 :1683-1690

Wani, G.M. 1995. Early diagnosis of pregnancy in surrogate mothers (Sheep & Goats). National Symposium on Embryo Transfer and Genetic Engineering Technologies in small Ruminants, May 7 – 8, CIRG, Makhdoom, Farah, India

Wani, G.M.2003. Biotechnological advances in goats production National Workshop on "Recent Advance in goat Rearing. MPKKV, Agric. Uni, Rahuri, 29-30 De, 2003

Wani,G.M. 2005. Advances in ovine & Caprine Reproduction www.Word-mavt. Com.Summaries free. Research paper 9 : 1-25

Wani, G.M.2006.
1. Development of Embryo and maternal interaction in small Ruminants. Synopsis free. Pages 13 price $ 50 words 1932
2. Advances in Ovine of Caprine Reproduction Synopsis free. Pages 25 Price 80 words 6725.
3. Development of Reproductive biotechnologies for sustainable livestock production synopsis free page 4 price $ 25 Words 870
4. Biotechnological advances in goat production synopsis free. Page 10 price $ 40 words 2700 Web. www.Work-mark.com.Research Papers.

Lee,K.Y. and De Mayo, F.J.2004. Animal models of implantation Reproduction. 128:679-695

Wiley, A.A; Bartol, F.F. and Barron, D.H.1987. Histogenesis of the ovine uterus. Journal of Animal Science. 64:1262-269

Spencer, T.E. Stagg, A.G; Joyce, M.M;Jenster, G;Wood, C.G; azer, W.F. Wiley, A.A. and Bartol, F.F. 1999. Discovery and Characterization of endometrial epithelial messenger ribonucleic acids using the ovine gland knok out model. Endocrinology, 140:4070-4080

Tamada, H; Tsubutani, D; Kawate, N; Inaba, T; Matsuya ma, S; Imakawa, K; Sakai, S; Christenson, R.K. and Sawada, T. 2002. Detection of transforming growth factor alpha and epidermal growth factor receptor RNA immunohistochemical localization of their proteins in the ovine uterus during early implantation period Histochemical localization of their proteins in the ovine uterus during early implantation period Histochemical Journal. 34:383-390

Mueller, M.D; Vigne,J.L; Minchenko, A;Lebovic, D.I, Leitman, Dc and Taylor, R.N. 2000. Regulation of vascular endothelial growth factor (VEGF) gene transcription by E2 receptors alpha and Beta. PNAS. 97: 10972-10977

Wang, W;Li, Q; Lin, H; Yu, X Qian, D; Dai, J; Daun, E and Zhu, C.2003. Expression of vascular endothelial growth factor and its receptors in the rhesus monkey (Muscaca mulatto) endomenterium and placenta during early pregnancy. Molecular Reproduction and Development. 65:123-131

Rowe,A.J; Wulff. C. and Fraser, H.M. 2003. Localization of MRNA for vascular endothelial growth factor (VEGF), angiopoitins and their receptors during peri-implantation period and early pregnancy in marmosets (Callitrix Jacchus) Reproduction. 126:227-238

Hilton, D.G. 1998. Leukemia inhibitory factor in sytokines, PP 277-296 ed. AR-Mirc-Sluis. New York, Academic press

Tulac, S;Nayak, N.R; Kao, L.C; Van Waes, M; Huang, J; lobo. S; Germyer; A, Lessey, B.A, Taylor, R.N, Suchanek, E and Giudice. L.C.2003. Identification, Characterization and regulation of the canonical Wnt segnalling pathway in human endometerium, J. Clinical, Endocrine and Metabolism 88:3860-3866

Dey.S.K.2004. Focus on implantation reproduction 128: 655-656

Wani, G.M. et al 2000. Effect of Oocyte harvesting techniques on in vitro maturation and in vitro fertilization in sheep. Small Ruminant Research 36.(1):63-67