New Generation Reproductive Technologies (stem Cell-nanotechnology)

I. Advances in Embryonic and Placental Development

1.Abstract:
Early embryonic development needs detailed investigations if we have to reduce embryonic mortality. Embryonic mortality details have been reviewed (wani, 2005). The reproductive abnormalities have been discussed and a sequential photographic presentation of embryonic mortality have recently been published (Khatoon et al 2007). The critical period when maximum embryonic losses go unnoticed is the period before attachment. Losses before actual union of foeto-maternal tissues can be missed and calculations of embryonic mortality estimates remain unaccounted for. Some recent studies of (Wani 2006, Khatoon et al 2006) have paved a new innovative model for investigation on foeto-maternal development and its attachment. This paper describes the detailed embryonic development, blastocyst elongation, formation of cotyledons within elongated blastocyst, their ramifications and networking of vasculature and formation of foetal placental unit. Conversely, a detailed photographic evidence of uterine receptivity, formation of a multicoated immunological barrier- the beginning of foeto- maternal- barrier mechanism along with caruncle formation, uterine milk formation and finally union of cotyledous with caruncles has been photographically documented.
2.Introduction:
Second and 3rd generation animal biotechnologies as reviewed (Wani, 2005) are multiple ovulation, embryo-transfer, preservation, cloning, micromanipulation splitting of embryos and nuclear transfer. The 4th Generation reproductive technologies have advance beyond our imagination and now we do have transgenic goats giving milk rich in insulin. (Wani 2007). The stem cell technology, embryonic cell culture, pronuclear micro-injection along with sperm-mediated, transgenesis have been in use and are precisely reviewed and discussed (Wani 2005, 2007).
The application of this advancement for diagnostic, therapeutic and economic uses especially its transfer to human medicine or for field application in animal industry needs further research and evaluation.
The another fact which can be of importance is the number of cells per embryos. During first day these may be two cells but after 8 days the cell number increases to 120 or more. It takes 32 hours for the oocyst after fertilization to reach the two cell embryo. Another 13 hours are needed to develop it into a four cell embryo. In another 15 hours 8 cell stages is reached. It is from here onwards that it takes 30 hours for a embryo to be converted into a Morula. This has been one of the most critical periods of embryonic development in ovine. A genematic diagram of various stages of follical maturation of oocytes development and further development has been reported (Wani 2005a) figure from web site is also show that the protein content after fertilization of 16 day old embryo increased by 7500 times. Morphologically the embryo size varies from spherical ovid to elongate after day 13th past coitus. New protein synthesis per unit of embryo is maximum during 8-13th day old embryos. Similarly phosphorylation per unit of embryo is also ten times more in 8 day old embryos than the later stages. Glucose and lactose production increased up to day 16th and the maximum metabolic weight is up to 14th day past coitus. For increased productive synthesis, aminoacid requirements are made, through production of allanic and glutonic acids by the embryos. The matobolic products get rid of amines. The signal transduction from maternal to foetal controls have been discussed (Moris et al 2001).
3. Dominant follicle dynamics
The new animal , products, medico-Veterinary packages, profitable genetic (trait) improved DNA Chips, more resistance giving gene make ups shall have a new role in future. For the next coming super advanced technology driven century, we must be prepared to use the 4th generation technologies for animal Welfare and Protective mechanisms. We thought and used Laparoscopy and ultrasonography 30 year ago (Wani et al, 1979, Wani 1981,1982), which has now become a “buzz” word and further sophisticated and modernized by my students across the world (Aggarwal, 2006, Wani, 2006). Our concept of DNA finger printing of cashmere goats in 1990’s (Khatlani et al, 1995) has now crossed the barriers of technology advancement. Today, in these pages, we shall be presenting our own results on
1. Embryonic development, formation of cotyledons, blactocyst development ramification of vascular system, net working of blood vessels to form initial placenta, their union and attachment processes. We shall describe morphological physiological, endocrinology, histochemical and receptor analysis to support our observations. This is 5th generation reproductive technology base for development of in-vitro-foeto models.
For 6th – 10th generation reproductive scientists we shall present theoretical hypothesis a vision -2050 in the form of.
2. Use of stem-cell, germ-cell and Nano- technologies for production of disease free, highly prolific reproductive gametes, their preservation and conservation. The use of these and other molecular and new-generation DNA-related bio-diagnostics shall also be discussed.

4. Uterine morphological changes:
The early pregnancy uterine morphological land marks were described only externally per rectum in bovine (Zamznis, 1976) some of its significant features have been summarized in a booklet (Wani, 1986). It describes easy palpable signs of early pregnancy in bovine and compares its observations with equines, sheep and goats. Some of the earliest indications of pregnancy by physical examination were:

i) Presence of corpus luteum of pregnancy early 15 day post-coitus
ii) Asymmetry of uterine horns 30-35 days post coitus
iii) Fluctuation around 40-49 days in cattle. Thereafter the advanced pregnancy signs like foetal membrance feel and slips. Palpation of placentomes and full foetal are known foetal membrane .
Various developments of foetal signs have been described (Wani, 1986) . The month wise morphological assessment of foetus in sheep has been reported (Mufti, 1995, Mufti et al, 1997) . Various reviews reports and books now describe events in development of foetus (Wani, 2005, 2006, 2007).
5. Development of conceptus-in vitro foetus
Pregnancy is an intimate association between mother and foetus. The protective covering on maternal caruncles and over wrap of foetal cotyledons with Blastocyst membranes, transformed into foetal membranes give the “Homograph” status to foetus. The mechanism of ruminant uteri has been discussed (Gray et 2002). Its more recent findings and attachments are the topic of one of our students.
We have tried to univel many obscure mechanism on blastocyst formation, plancentation and development of cotyledons. The protective mucin coverings over uterine caruncles and their union with cotyledons has been snaped.
The internal changes have been correlated with biochemical, morphological and histochemical and histoanatomic feature of the gravid and non-gravid uteri.
6. Foeto-maternal interaction:
The estrogen, progesterone in foetal and maternal blood have been estimated along with estrogen progesterone receptor concentrations in the pregnancy and non pregnant states.
Endometrial receptivity has been discussed (Huct-Hudson, et al 1989, Wani 1996, Wani, 2003, 205, 2006). Proliferation of uterine cells is influenced by E2 and P4. These hormones have their receptors in uteri called as E2 (ER) , P4 (PR) receptors (Lee and Demyo 2004) we have also investigated the same and are being analysed. Our studies indicate certain changes which project a new mechanism of foeto-maternal unions (Khatoon and Wani, 2007).
Various growth factors described (Wani 2006) may be helpful in future for invitro-foetal growth.
7. Reproductive management
The reproductive management strategies to enhance meat production could safely be divided into two kinds:
i. Traditional management: Animal are still managed under zero- input/ migratory /tribal or extensive management system. The application of above described methods will be limited. They may indirectly help in this system by better sire availability.
ii. Modern management: Zero-grazing, stall-fed or semi-intensive, commercial goat farming system may use the above methods more and more to produce high quality germplasm. They may use variety of improved reproductive technique or reported (Wani,1996; Wani et al., 200a; Wani et al., 1998; Mufti et al., 1997; Wani and Sahani, 1981; Wani et al., 1986, 1987, 1988; Wani and Buchoo, 1991; Buchoo and Wani, 1991; Wani, 1989, Wani, 1995; Wani, 1984a and b; Wani and Sahani, 1980a).
8. Enhancement of fertility
Desired fertility objectives have been discussed (Wani, 2001). Increased prolificacy, early rebreeding, safe/clean parturition, set-time animal crop, elite gene introduction are some of the desired fertility enhancement goals. The constraints in achieving desired objectives in small ruminant livestock sector are the problems of inbreeding, reproductive losses (Wani, et al., 1980; 1981; 1994), pre-pubertal mating, indiscriminate or illicit mating, venereal diseases, nutritional and other stresses. These above referred factors reduce fertility status of our ruminants especially under transhumane migratory systems.
These constraints mentioned could overcome by adopting enhancement strategies (Wani, 2001), which include exchange of sires by tribal, Gujjar, Bakarwals and Chopans to reduce inbreeding. This could be ensured through legislature or forced castrations and introduction of sires from outside the flock. The pregnancy detection methods described (Wani, 1981) and later on used extensive in J&K under farm condition (Wani et al., 1998; Wani, 1989) needs adoption as a routine in each flock for better care of offspring and dam.
9. Corpora luteal biosensitivity.

Corpus luteum growth and development is the first earliest pregnancy signal along with maternal recognition factors discussed (Wani, 1996). The corpus luteum development and receptivity to PGF2 alpha is now known. The ascending and descending corpora lutea are insensitive to PGF2 alpha. What makes them insensitive and what resurges their sensitivity need exploration. May be in future, CL insensitivity might have direct effects on survival of the conceptus. Does conceptus make CL more sensitive to PGF2 alpha? Or in other words, does the gravid CL and non-gravid CL behave similarly or differently to PGF2 alpha. Many such questions vis-à-vis the ovarian morphology, uterine environment and behavioral patterns need to be studied and co-related to develop a manipulative and curative safe system. In some species CL of pregnancy is necessary till delivery, in others it is needed only till placenta develops. Its complete genesis and biostimulation and bioregulatory roles are obscure and needs further research.
10. Dominant follicle dynamics

The dominant follicle dynamics may be in focus for ovulation control. Dominant follicle or would be bride at each ovulation appears around day 7 of cycle in women. It occurs much earlier on day- 1 to 0 in ruminants (cow, sheep,goats). This time point may be the focus to try future selective interferences in ovulation control. Antagonist use during this phase has shown decreasing levels of estrogens in serum thereby indirectly hinting at dominant follicle alteration, as it is one of chief source of this hormone during this phase (Fraser, 1987). Dominant follicle in its sojourn to freedom and release at ovulation seems to attain some specific qualities. Why? It alone releases itself from bondages in ovary (ovulation) and rest of its competitors are hindered through the process as atretic follicles. What make this dominant follicle to attain these qualities among a vast pool of follicles needs further study. In coming 50 years one shall possibly understand and interlock a pre-determined messenger system triggering dominance in one or few follicles destined to become the new offspring of that species. This system when decoded shall offer new opportunities in controlling ovulation both for contraception (women) or augmentation (animals). New genepool of dominant follicles from dead high yielding animal ovaries would help to propagate and multiply the elite.

Over stimulatory effect during superovulation could be avoided by use of antagonists, as dominant follicles seem to evade suppression, thus follicular selection will prevent excessive stimulation and cystic follicles. Follicular fluid constituents containing occyte awaiting for biochemical signals to resume (meiosis ) growth and development to ensure timely and healthy ovulation need more exploration (Lenton et al., 1988). The specific signals triggering occyte release might help in in vitro occyte maturation success rate.

11. Development of conceptus-in vitro foetus

The lean meat production needs more studies on development of conceptus. The prenatal development undergoes in three stages. The early pregnancy signals, skeletal & nervous tissue growth, on priority. During mid-gestation the hind and fore-limbs develop. During late gestation muscular development along with peripheral extremities including hair develop. In order to increase lean meat production, research in needed to investigate relations between hard and soft tissues. Maternal and foetal interactions needs a probe. Some basic studies (Mufti, 1996) indicated prospects of manipulating lean meat production (Wani, 2001, 2002 a) by bio-stimulators during late gestation, which may help, the production of muscular lambs/kids at birth, besides, increasing birth weight. More details have been described (Wani, 2001, 2002a) Fig.1-5). More studies in future are needed to reveal the exact mechanism involved in critical transition of power control from maternal to embryonic genome. Application of recent techniques of reverse transcription, PCR and others may reveal the details (Schultz et al., 1990).

12. Slaughter house follicle pool

A good number of our endangered species die where climatic conditions are hard and harsh. Under temperate climatic conditions dead bodies are entrapped in snow for long. It gives a hope to revive their follicular pool. Our studies on resumption of life using follicles from slaughtered sheep and fertilizing them with epididymal semen was an pioneering attempt in this direction (Wani, 1996) (Fig 6-8). The protein content of unfertilized ovum is around 100 mg in rabbit, 27 mg in mouse (Brinister, 1967, 1971). The small genetic material needs extensive analysis to unveil vast informations in its codans and anticodans. Further ovum maturation or resumption of meiosis is initiated during birth or shortly before birth and thus the pool of follicles remains fixed at birth. Therefore, foetal ovary has to be the subject of further manipulations especially in view of its immunological insensitivity (Guraya, 1985).
13. Gamete biosafety

Bacterial counts in semen in unprecedented numbers effect semen fertility (Kher, and Dholokia, 1985), The contaminational hazards have increased due to environmental pollution, water and air contamination. Above all the development of resistance of known bateria and other micro-organism to antibiotics (Gupta and Maurya, 1993). We have examined the semen of about 40 bulls maintained at the Frozen semen bank, Rambirbag, Kashmir for bacterial loads in semen and tested antibiotic sensitivity (Koul, 2002).

Presence of bacteria in large numbers in semen, effects its quality and fertility (Kher and Dholokia, 1985). The source of contamination of semen vary from inflammatory foci and lesions on penile apparatus, contaminations on equipments, appliances, diluters, extenders, air and laboratory environment. These contamination hazards have increased with environmental pollution. Many bacteria having developed resistance to routine antibiotics used (Gupta and Maurya 1993) in semen extenders has made inseminations more critical and vulnerable. More studies in this direction are needed to develop a bio-safe and infection free germplasm pool of frozen and fresh semen in future.

14. Foeto-maternal interaction

The role of endometrial stromal cells during pre-implantation stage needs further research. Some studies on ultrastructure of endometrium (Brinsfield et al., 1974) are still not sufficient to answer the questions. What is the role of immune cells and endometrial cells in building of the immunological barrier to protect the foetus. Foetal biology is abarren field of research in the world. After few initial steps by Dr. Moris Young in UK and D. Srivastava in India, only scattered information is available on the subject (Wani and Buchoo, 1990; Mufti, 1996) . Much is still to be unveiled by 2050. The placental hormone influence mammary development and milk production of the dam. Foetal membranes serve as Chief source of nutritive exchanges. The arrangement of cotyledons, caruncles, positioning of single and twin conceptus, indicate complex mechanisms with common roots. (Mufti et al., 2000) These mechanisms of foeto-maternal interactions and relationships emphasize proper foetal number diagnosis and corresponding materno-nutritional management strategies.

15. Nanotechnology:

In the proceeding pages we have seen the role of biotechnology in animal reproduction. The coming generations will harvest the gains in the form of more micron-level-accurate technology being replaced with nanogram-level-invisible technology- the nanotechnology. Now even converging technologies are shaped resulting from merger of Nano- science and Nanotechnology with biomedicine, bio-sciences, biotechnology, genetic engineering and information technology.
The cognitive sciences like Neuro- sciences, computing, communication combined with nanotechnology may be so mystic and deterministic that science shows will appear as “Majic shows” . This new technology wave” may define our spiritualistic wavelengths. Many unsolved or resolved facts on birth and genetic make up shall send such messages as was known of Sagic saints, peers and Fakirs.

The birth of a technology needs patience of public and policy makers. In the past “Michael Farady” – the inventor of electric generator, was asked by the then Chancellor of Exchequer, as to what use is the electricity you are generating. He argues but no argument convinced the accountant. Finally, he promised him that the day will come when you can tax it. Today a remote destination on this earth demands 24 hr electricity. They never argue its price, but its availability as savoir to life be it medical, veterinary or any other field. Thus like Farady I profess and advocate experiments on use of nanotechnology in animal reproduction today. It may sound strange, but yes in my life I shall fore see a role of nanotechnology in Animal reproduction.

16. What is Nanotechnology:

Technology related to features of nanometer scale. Thus we will unveil gametes in minute details; sperm head colliding zona-pellucida shall be filmed. The radial blast of cortical reaction shall be known. All chemical messengers shall be unveiled at nano-level.
The shape of gamete, Zygote, embryo and foetal development at nanolevel shall bring forth new additions to our knowledge. The use of such information through use of computer languages, both hard and soft shall make this knowledge dispensable and useable at remote-corners of the Universe. Combing cognitive, mythical and mysterious happening within womb could be registered and translated via nano-ultra-sonography.
Thus futuristic view of nanotechnology will be nanographic follow of information and has been described ( Mordini, E, 2007).
Nanotechnology may allow us to send tiny nano microprocessors into the animal womb to send us signals which we wish to know for prevention of diseases. The stem-cell-germ-cell technology combine with Nanotechnology shall bring new hope and future for the propapagation of Gene-technology-ETT- A-I Combine. It may sound like sending a wave of spirit to fertilize an “egg” – thus proving the “God Word in Quran of creating the Chirst through mystic spirit. This and other convergence of known reproductive technologies are challenge to us in future
Nanaoparticels developed can be agents which would use blood and carry durgs for curing disease at molecular levels. Thus a new mechanism of targeting infective agents of smaller dimension could be a possibility.
17. Stem-cell-Technology:
Stem- cells are undifferentiated cells. The embryo at morula stages have undifferentiated blastomeres. These cells can transform themselves into 200 or more cell type, which could be used to repair or regenerate new desired cells. This advanced cell research may help diseased people like those suffering from cancer, Alzheimer’s disease syndrome, Parkinsons disease and even paralysis.
The stem cells are derived from 4-5 day old embryos or fertilized cells. The stem-cell-technology has a new role to play in Animal reproduction.
Firstly, the stem-cell source could be pooled out of slaughter-house oocytes or from the vast pool of embryos hatching out in many animal species.
Secondly, the cell source could be obtained from foetal culture and if new-grown-immunoprotectant cell of foetus could help or provide germ-cell specific to any particular organ, the repair of organs could be a new revolution.