oogenesis - human ovary

OOGENESIS - HUMAN OVARY A pictorial presentation of images Fine Structure of Oogenesis & Folliculogenesis in the Fertile & Infertile ovary

Kamala Selvaraj & Henry Sathananthan Dedicated to late Prof P.M.Motta (Rome) Monash Institute of Reproduction & Development Melbourne Australia GG Hospital Chennai, India Copyrights reserved

DEDICATION TO PROF. P. M. MOTTA (1942-2002) This CD-rom Is Dedicated To The Memory of Prof. Pietro M. Motta, Rome Dr. Pietro M. Motta, Professor of Anatomy, Rome was born in Sassari (Italy) on September 18th, 1942. He graduated obtaining Ph.D and M.D degrees at the University of Messina, Italy and was appointed full Professor of Anatomy, Faculty of Medicine, University of Roma “La Sapienza” and became Director and Chairman of the Department and Member, Senatus Academicus, very prestigious appointments. He was Past-President of the Italian Society of Anatomy, General Secretary of the International Symposia on Morphological Sciences, President of the International Federation of Associations of Anatomists and Honorary Member of 16 International Scientific Societies, including the American Association of Anatomists. Pietro specialized in the Biology of Reproduction, especially Ovary, and published 350 papers; 50 invited articles in micro- and macro- anatomy in edited journals and in 37 books as Author/Co-author/ Editor/Co-editor. He held 6 Medical Doctorates “Honoris Causa” in various universities and was an active member in 20 professional societies. He was on the Editorial Board of 27 national and international journals, of repute. Prof. Motta was a consultant for the Italian Medical Dictionary (Encyclopedia Medica Italiana) for which he also prepared several chapters. He has also collaborated as scientific consultant to various TV programs related to Anatomy in the Series "Quark" for Italian radio and television ( 1985-1990, 1993-1996 ) His exquisite diagrams and illustrations are reproduced in several textbooks and atlases of Anatomy, Histology and Embryology and many popular reviews and journals all over the world, including this CD-ROM. Some outstanding pictures and works are permanently exhibited in international museums and he received numerous scientific international awards. He was a great teacher and research director and had a charismatic, amiable personality. Pietro was President and organizer of 4 main international Congresses: VIII Intern. Symposium on Morphological Sciences (Rome, Italy, 1988); 2nd Intern. Malpighi Symposium (Rome, Italy, 1995); 3rd Intern. Malpighi Symposium (California, U.S.A., 1997), XV Congress of Intern. Association of Anatomists and 4 th Intern. Malpighi Symposium (Rome, Italy, 1999), to 3 of which Dr. A.H.Sathananthan was invited. We take great pleasure in dedicating this visual presentation to our dear friend and mentor Prof. Pietro Motta, a great scientist of the twentieth century.

AUTHOR DR.KAMALA SELVARAJ Dr. Kamala Selvaraj is Associate Director, GG Hospital, Chennai, India. She is the daughter of Tamil actor, Gemini Ganesan and is an Obstetrician and a Gynecologist. Situated in the heart of the city of Chennai is her dream project "Fertility Research Centre” a pioneering center in Assisted Reproductive Technology with a well equipped IVF/ART laboratory. After graduating from Kasturba Medical College, Manipal she completed her DGO and then her MD from Madras Medical College, Chennai. She then went for training in Invitro Fertilization and Embryo Transfer in Monash University in 1985 and attended several training programs in Singapore and Monash University. Dr Kamala commissioned the First Test Tube baby of South India in August 1990 and since then has had many achievements to her credit. Twin babies were born to a patient with Mayer-RokitanskyKuster-Hauser syndrome, thus giving hope to those with no uterus, the first of its kind in Asia. She has also published many papers in National and International journals and has received many awards for her outstanding achievements in the field of Assisted Reproduction. She received the “Seva Ratna” Award instituted by the Centenarian Trust in the presence of His Holiness Sri Jayendra Saraswati and Best Lady Doctor Award in 1993. She has recently been awarded Ph.D for her thesis on “Premature Ovarian Failure and its Management which is a widely discussed subject. So far, the numbers of pregnancies by IUI are nearing 6000 while pregnancies by ART has exceeded 900 in number. www.gghospital.in

AUTHOR PROF.A.HENRY SATHANANTHAN Dr. A. Henry Sathananthan is one of the pioneers in IVF and assisted reproductive technology (ART) in Melbourne, Australia and has worked with the Monash Team (Obstetrics & Gynaecology and Center of Early Human Development), since 1977. He has also been involved in collaborative research with the Department of Obstetrics & Gynaecology at K.K. Hospital & The National University Hospital, Singapore since 1983, who pioneered IVF in Singapore. Currently, Dr. Sathananthan is an Honorary Associate Professor of the Monash Institute of Reproduction & Development & Adjunct Professor in Monash Immonology & Stem Cell Laboratory, Monash University, after his retirement as Associate Professor & Reader of Latrobe University, Melbourne. He is now Honorary Adjunct Professor, Anatomy & Developmental Biology, Medical Faculty, Monash University, having completed 37 years Honorary work at Monash University. He was also a Research Professor and consultant in ART in two hospitals in Chennai and Bangalore, India. He has over 156 international publications, including 95 refereed papers, reviews, 50 chapters, 4 atlases and 8 CD-ROMS and over 110 conference & workshop presentations. He has also been CD-ROM editor on the Editorial Board of Human Reproduction Update, Cambridge (1995 - 2000), the most highly rated journal (impact factor ratings) in Obstetrics & Gynaecology and Reproductive Biology in 2000. Henry has his own website www.sathembryoart.com Dr. Sathananthan has a Ph.D in Embryology (Reading University, U.K.) and has had 53 years of teaching experience in micro-anatomy at all university levels, having graduated from the University of Ceylon in 1959. He has specialized in using illustrations, audio-visuals, CD-ROMs, atlases and the web for both teaching and research. He is also a visiting professor in Sri Lanka, having taught in several universities and is advisor to the Minister of Higher Education. He was trained in electron microscopy at Oxford University by the Late Prof. John R Baker, FRS. Dr. Sathananthan is a world authority on human fertilization and early embryogenesis and his most significant discovery is the inheritance, replication and perpetuation of the human paternal centrosome in 1991 and its impact on infertility. He is now contemplating to publish his images on Epub for posterity.

BACKGROUNDS OF CLINICIANS Dr. Priya Selvaraj, obtained her masters degree in Obstetrics and Gynaecology (1997- 2000) from Sri Rama Chandra Medical College and Research Institute, and is presently attached to Fertility Research Centre (FRC), GG Hospital, Chennai. She has had IVF & ICSI training in National University Hospital Singapore apart from Endoscopic surgery training at Keil, Germany and National University Hospital, Singapore. She has actively taken part in the CME programs conducted by the hospital and has attended national IVF Conferences. She is now Medical Director of GG Hospital, doing clinical IVF & ART with her mother Kamala.

Dr. Vijaya Ganesh, graduated from Madras Medical College (1982- 1986), Chennai and since then has been attached to Fertility Research Centre for over a decade. She is trained in IVF & ICSI at National University Hospital, Singapore. She has attended several national and international conferences. She has participated in conducting IVF workshops, CME programs & panel discussions organized by Fertility Research Centre, GG Hospital, and Chennai. Presently she is actively involved in the IVF department, FRC, GG Hospital, Chennai.

Dr. M.S.Lakshmi has a MBBS DNB in Obstetrics & Gynaecology and is now IVF coordinator at the ART Center in Coimbatore, India and was a clinician at Fertility Research Center, GG Hospital for over 10 years. She followed a workshop in ART at NUH Singapore and has specialized in Ultrasound, Laparoscopy & Hysteroscopy, Infertlity Management and ART techniques. She has presented papers in conferences in India and attended several conferences and workshops.

OVARIES EXAMINED (RELEVANCE) • 3 FOETAL OVARIES AFTER TOP (13-15 WEEKS OF GESTATION) (Primordial germ cell ‘PGC’ technology & Oogonia)

• 3 FERTILE OVARIES OF WOMEN WHO CONCEIVED (AGES 27,30,32) (Controls for follicle & ovarian tissue freezing, banking & culture)

• 3 PCOD PATIENTS CONFIRMED BY ULTRASOUND (AGES 26,28,31) (In vitro maturation of oocytes and culture for ART)

• 3 POF PATIENTS - ANOVULATORY (AGES 28, 32, 35) (Treatment of patients – oocyte donation)

(MOST OF THE IMAGES PRESENTED IN THIS CD ARE ORIGINAL)

INTRODUCTION & AIMS - OVARY STRUCTURE The ovary , the female gonad, is a highly complex organ both structurally and physiologically, with a variety of somatic cells and, of course, follicles containing oocytes in various stages of development, ranging from primordial, growing to mature antral follicles. It is amply supplied with connective tissue, blood vessels, nerves and smooth muscle and is surrounded by a surface (coelomic) epithelium. Its stroma is differentiated into a cortex (with developing follicles) and a medullar (with blood vessels). The ovary is a major endocrine organ interacting with the pituitary gland and the uterus during each menstrual cycle and is regulated by gonadotrophins (FSH and LH) and secretes steroid hormones- estrogen and progesterone, which interact with the endometrium (see texts, Larsen, 1998; Moore, 1982).The various stages of folliculogenesis and oogenesis are presented in these texts and in numerous publications by Prof. P. M. Motta and his Colleagues in Rome. Some relevant publications and reviews are cited at the end of this presentation (Motta et al.1994, 1995; Gougeon,1997: Wartenburg,1989) The human oocyte, the female germ cell, shows progressive changes in fine structure during folliculogenesis and oogenesis until it is ovulated and ready for fertilization. Parallel changes also occur in its relationship with the surrounding follicle cells, which support and nurse the growing and maturing oocytes till ovulation. The vast majority of oocytes (~7 million) are lost, undergoing atresia, during the life time of a woman from foetus to sexual maturity and only about 400 – 500 oocytes are normally ovulated from puberty to menopause. Apoptosis, programmed cell death, is believed to be a cause (De Pol et al.1997). Hence the vast majority of oocytes and follicles are destined to undergo atresisa. Atlases portraying the structure of oocytes have been published (Sathananthan et al.1993,1996; Veeck,1999). Some images are also on the web www.sathembryoart.com This CD-ROM is an original, concise, visual presentation of the fine structure of the human ovary in health and disease. We will cover the structure of cortical ovarian biopsies of young fertile and infertile women obtained by informed consent, a research speciality of Dr. K. Selvaraj and her clinicians. We also present some images of foetal ovaries and pre-ovulatory oocyte maturation in vitro, to complete the story of oogenesis . This study of the ovary is important and topical, since ovarian follicles and tissue are now been frozen and cultured (Hartshorne,1997;Shaw et al.1997;Hovatta et al.1999), whilst ovarian tissue banking is now a reality. Furthermore, in vitro maturation (IVM) and freezing of oocytes is currently being researched (Trounson et al.1998) and polycystic ovaries are been used for this purpose. We present the structure of ovaries of polycystic (PCOD) patients (Franks,1995), as well as, those of premature ovarian failure (POF) patients ( Selvaraj “Premature Ovarian Failure & its Management” Ph. D. Thesis; Vegetti et al.1998) who are mostly anovulatory, to help them conceive after ART or by oocyte donation. We present over 70 images of the human ovary (foetal and adult), photographed by digital light microscopy (LM) and transmission electron microscopy (TEM), which include some explanatory diagrams, edited by Photo Shop 6 & Paint Shop Pro 5 on power point. The pictures are fully labelled with concise footnotes and should be an invaluable resource to patients, students, teachers, clinicians, researchers and technicians involved in assisted reproduction . ONLY ORIGINAL MAGNIFICATIONS AT MICRO-PHOTOGRAPHY ARE INDICATED IN FOOT-NOTES

FOLLICLES, OOCYTES & MATURATION - MEIOSIS

FOLLICULOGENESIS OOGENESIS (Courtesy Veeck,1999)

OVARY (PCOD) - ENDOSCOPY

The 2 pear-shaped ovaries (whitish) & ligaments are evident in the body cavity

OVARIAN BIOPSIES & TEM - METHODOLOGY Ovarian Biopsies were done by operative laparoscopy or during hysterectomy following method for Polycystic ovaries. PCOD was diagnosed by ultrasonographic appearance of at least ten peripherally oriented multiple cysts of approximately 8 to 10mm (usually >9mm) in diameter surrounded by dense stroma. The stroma is usually about twice the diameter of the pericortical cystic area and should occupy >25% of the ovarian volume. Three fertile women (ages 27, 30,32), 3 PCOD patients (26,28,31) and 3 POF patients (Selvaraj,Ph.D. thesis) were selected for biopsies. Three foetal ovaries (13-15 weeks) were obtained after TOP and examined, as described previously (Sathananthan et al.2000). Only if the patient was found to be medically fit, she was subjected to operative laparoscopy. The patient was asked to empty the bladder before the procedure. Under general anaesthesia, patient was put in 1 ithotomic position, pneumo peritoneum was created using Verees needle inserted through a vertical incision within the umbilicus. Once appropriate pneumo peritoneum (18 to 20mm Hg) was achieved Verees needle was removed and 11 mm Endo tip was inserted. The patient was then replaced in the Trendelenburg position and two side ports were inserted. Pelvis and its contents were examined systematically. The ovary was held with a non-tooth forceps, using a rounded scissor and cortical biopsies were taken from the ovary. Haemostasis was secured using bipolar cautery. All biopsies were immediately placed in fixative and stored for TEM. Ovarian biopsies were fixed routinely in 3% Glutaraldehyde in 0.1 M Cacodylate buffer (pH 7.3), postfixed in 1% aqueous Osmium tetroxide and rapidly processed in Epon or Araldite. Thick survey sections were stained with 1% Toluidine blue in Borax and thins were stained in alcoholic Uranyl acetate / Reynold’s lead citrate and examined by Leitz Digital and Joel electron microscopes, respectively. Images were edited and some were coloured with Adobe Photoshop 6 and Paint Shop Pro 5 and presented on Power Point.

EVOLUTION OF THE OOCYTE IN THE OVARY

Primordial germ cell to the mature oocyte is depicted including changes in mitochondria. (Reproduced from Motta et al, 2000)

FOETAL OVARIES (N=3) - OOGONIA Foetal ovaries are now being cultured or frozen, to generate oocytes for assisted reproduction, an emerging technology. We examined the ultrastructure of oogonia at 13-15 weeks of gestation which could be used as a control for culture and freezing of foetal ovaries for ART (Hartshorne,1997;Sathananthan et al,2000). Oogonia are largely located in the ovarian cortex, whilst primordial germ cells (PGC) and somatic follicle cells compose the surface epithelium. Oogonia are derived from PGC after they migrate to the ovary from the wall of the yolk sac in the 3rd week of development via the genital ridges. Oogonia and PGC have large vesicular nuclei with clear cytoplasm, compared to denser follicle cells which have polymorphic nuclei. Follicle cells intermingle with oogonia and establish close contacts at the beginning of folliculogenesis. Nuclei of oogonia contain 1-3 highly reticulated nucleoli, reflecting high levels of RNA synthesis at the onset of growth. Rough endoplasmic reticulum form stacks of cisternae associated with numerous ribosomes. Prominent organelles in the ooplasm are elongated mitochondria with dense matrices and tubular cristae presenting a multilocular appearance. Typical Golgi complexes, dense bodies and clear vacuoles are present and microfilaments are located beneath the plasma membrane. The origins of Balbiani Bodies (see follicles) can be traced to oogonial centrosomes that nucleate cellular organelles close to nuclei. The most remarkable feature of oogonia is that they have 2 typical juxtanuclear centrioles (diplosomes) with dense pericentriolar material which nucleate microtubules, characteristic of functional centrosomes. This is quite unlike mature oocytes but resemble centrosomes organizing the cytoskeleton in somatic cells. The mature oocyte has no centrioles, since the maternal centrosome is inactivated or reduced during oogenesis, while the paternal is dominant. This reduction is important, since only one centrosome need be functional in either gamete to ensure normal fertilization (Sathananthan et al.1993;2002) Centrioles are involved in mitosis of oogonia, before they mature into oocytes at the onset of meiosis. Oogonia are also important in the context of primordial germ cell (PGC) technology, like embryonic stem cells.

MIGRATING PRIMORDIAL GERM CELL IN EMBRYO

Microfilaments

Protrusion

Nucleoli

Nucleus RER

Six week embryo showing fusiform PGC, which arise from the dorsal wall of the yolk sack in week 3. x5800 (from Motta, 1995 )

FOETAL OVARY (15 WEEKS) – OOGONIA (LM)

Stroma

Epithelium

Several oogonia (purple) are nesting within the ovary. The epithelium and stroma are evident x100

FOETAL OVARY – EPITHELIUM & OOGONIA (LM)

Follicle cells

Oogonia

Nucleus

Coelomic Epithelium

The epithelium is multilayered (stratified) . The oogonia arise from PGC in week 9 and have large nuclei with nucleoli x1000

FOETAL OVARY – COELOMIC EPITHELIUM (TEM)

Light cells Dark cell Epithelium

Oogonium

The epithelium is stratified and consists of light and dark cells. Note degenerating cell (arrow) x2000

FOETAL OVARY – EPITHELIUM & OOGONIUM(TEM) Epithelium

Dark cells

Light cells Oogonium

Oogonium

Epithelium consists of light and dark cells. Note superficial oogonium surrounded by potential follicle cells x2500

FOETAL OVARY – NEST OF OOGONIA (LM)

Oogonia

Follicle cells

Lighter oogonia are separated by darker , potential follicle cells. Oogonia have large nuclei and few cytoplasmic organelles. x1000

FOETAL OVARY – NEST OF OOGONIA (TEM) Oogonium

Nucleus

Follicle cells Nucleolus

Oogonia have large nuclei and few organelles.Nucleoli are reticulated and mitochondria dark.(Sathananthan et al, 2000) x2000

FOETAL OVARY – SINGLE OOGONIUM (TEM)

Nucleus

Mitochondria Nucleolus

Follicle cell

The oogonium is surrounded by potential follicle cells. Note large nucleus with reticulated nucleoli. x5000

TWO OOGONIA SURROUNDED BY FOLLICLE CELLS

Follicle cells

A cell junction is evident between oogonia. R=RER G=Golgi. M=Mitochondria.x3000 (Sathananthan et al,2000) x3000

FOETAL OVARY(15 WEEKS) – TWO OOGONIA (TEM)

Follicle cells

Nucleus

Nucleolus RER Mitochondria

The two oogonia have an intervening cell junction consisting of primitive desmosomes (arrow) x4000

DESMOSOME BETWEEN FOLLICLE CELL & OOGONIUM

Oogonium

Follicle cell

Two desmosomes (arrows) are seen in the primitive cell junction between the 2 cells. x20,000

OOGONIUM – NUCLEUS & MITOCHONDRIA (TEM) Nucleus

Chromatin Nucleolus Mitochondria

The large nucleus has a reticulated nucleolus and peripheral chromatin. Mitochondria have tubular cristae x20,000

STROMA IN FOETAL OVARY (15 WEEKS)

Stromal cells

Stroma consists of connective tissue cells with few collagen fibrils and blood capillaries (not shown). x2000

BLOOD CAPILLARY IN STROMA-FOETAL OVARY

Stroma Capillary

RBC Collagen

Endothelium

The capillary has red blood cells surrounded by an endothelium. Note few collagen microfibrils. x2500

CENTROSOME WITH TWO CENTRIOLES IN OOGONIUM Follicle cell

Oogonium

Oogonia have typical centrosomes with 2 centrioles(arrow) , unlike mature oocytes, and resemble somatic cells. x6000

TWO CENTRIOLES IN CENTROSOME - OOGONIUM

PCM

RER

Dense pericentriolar material (PCM) surround centrioles which are barrel shaped and show triplets of microtubules. x100,000

CENTROSOME ORGANIZING CELL ORGANELLES Golgi Mitochondrion

RER

Microfilaments Centriole Cortical granule Microtubules

The centrosome (centriole) organizes the cytoskeleton and the spatial arrangement of organelles. (courtesy Prof P M Motta)

CENTROSOME ORGANIZING CELL ORGANELLES Nucleus

Centriole

RER

Microtubule

Rough endoplasmic reticulum (RER) and fine microtubules radiate from the centrosome in an oogonium. x70,000

OOGONIUM SHOWING ORIGINS OF BALBIANI BODY

Mitochondria

Golgi

Nucleus

BB

RER

The Balbiani body (BB) is an aggregation of cell organelles close to the nucleus. x10,000

OOGONIUM AT PROPHASE OF MITOSIS

Follicle cells Chromosomes

Nucleus

Oogonium

The chromosomes are decondensing within the nucleus. Oogonia initiate meiosis at about 13 weeks and arrest at prophase. x3000

OOGONIUM AT METAPHASE OF MITOSIS

Follicle cells Oogonium Chromosomes

Chromosomes have condensed on a spindle, which is organized by centrioles (not seen) . Nucleus has disorganized. x4000

FINE STRUCTURE OF FOLLICULAR OOCYTES

The arrangement of organelles are depicted in primary, secondary and growing oocytes. (Courtesy Prof P.M. Motta, Rome)

FERTILE OVARY (27 YEARS) - EPITHELIUM & STROMA Cortex

Stroma

Capillary

Epithelium

Epithelium is cuboidal and forms a single layer. No follicles are present in the cortex. x400

FERTILE OVARY (27 YEARS) - EPITHELIUM & STROMA Capillary

Epithelium

Stroma

Dark cell

Cortex Light cell

The cuboidal epithelium has two types of cells - light and dark Stroma consists of fibrous connective tissue. x1000

FERTILE OVARY (30 YEARS) – MEDULLA Capillary

Arteriole

Venule

Stroma

Blood vessels consist of arterioles, venules and capillaries in the central stroma x400

FERTILE OVARY (27 YEARS) – PRIMORDIAL FOLLICLE

Follicle cells Light cell

Stroma Nucleus

Balbiani body Oocyte

The follicle has a single layer of cells surrounding this diplotene oocyte. Note stroma surrounding follicle. x1000

FERTILE OVARY (27 YEARS) - PRIMARY FOLLICLE Basal lamina Balbiani body

Dark cell Nucleus Light cell

Oocyte

Theca folliculi Stroma

The follicle has 2 to 3 layers of cells resting on a basal lamina. The oocyte shows vacuoles-early atresia. x1000

FERTILE OVARY (27 YEARS) – GRAAFIAN FOLLICLE Epithelium Stroma Cortex Granulosa Oocyte Follicle

Medulla

Early Graafian follicle shows developing antrum and a multilayered granulosa. Note oocyte in cumulus. x50

FERTILE OVARY (27 YEARS) – GRAAFIAN FOLLICLE Granulosa

Theca

Antrum Zona

Corona radiata Oocyte

Cumulus

Early Graafian follicle has an antrum and a secondary oocyte about to intiate 1st.meiotic maturation prior to ovulation x200

GRAAFIAN FOLLICLE – SECONDARY OOCYTE Antrum Nucleus Zona Corona

Oocyte

Cumulus

Granulosa

The oocyte is clearly surrounded by the zona pellucida and corona cells. Note nucleus in oocyte and fluid filled antrum. x400

GRAAFIAN FOLLICLE – SECONDARY OOCYTE (TEM)

Corona cells

Cortical granules

Zona RER

Oocyte

The oocyte has a single layer of cortical granules beneath the surface, surrounded by the zona and corona cells. x2000

RELATIONSHIP BETWEEN FOLLICLE CELLS & OOCYTE

Three dimensional relationship between follicle cells and oocyte in a mature Graafian follicle, showing gap junctions and desmosomes. (Reproduced from Motta et al, 1994)

GRAAFIAN FOLLICLE – CORONA CELL JUNCTION Zona

Cortical granule

Cell process Desmosome

RER Gap junction Oocyte

The corona cell process ends in a junction in oocyte - shows 2 desmosomes and possibly 2 gap junctions ? (arrows) x20,000

POLYCYSTIC OVARIES – PATIENTS (N=3) CASE I ( PCOD ): A 28 year old lady reported to our hospital with history of primary infertility. She attained menarche at the age of 14 yrs and had irregular menstrual cycles. Diagnostic laparoscopy revealed normal uterus, normal ovaries and normal left fallopian tube with beaded right fallopian tube and was found to be anovulatory. Hysterosalpingogram revealed normal uterus with bilateral spill. Her basal hormone levels recorded at our center were: FSH=10.40 mIU/ml, LH=14.84 mIU/ml, Prolactin =8.50 Ng/ml, T3=1.42ng/ml, T4=10.7 g/dl, TSH=3.16 IU/mL and free T4 = 1.03 ng/dl. Her blood sugar level was found to be 190 mgs/dl. The patient was found to have mild Diabetes Mellitus while on Metformin. Her husband’s seminal analysis was found to be normal. Transvaginal ultrasonogram showed PCO and the patient was found to be hypothyroidic. CASE II ( PCOD ): A 31 yr old lady came to our hospital with primary infertility. She attained menarche at the age of 13 and had irregular menstrual cycles. The patient was obese and was found to be diabetic and has undergone treatment for thyroid disorder. Diagnostic laparoscopy revealed normal uterus, normal ovaries with patent fallopian tubes. Her basal hormone levels recorded at our center were: FSH=5.06 mIU/ml, LH=6.59 mIU/ml, Prolactin =12.85 Ng/ml, T3=3.82ng/ml, T4=19.9 g/dl, TSH= < 0.05 IU/mL and free T4 = 3.19 ng/dl. The transvaginal ultrasonogram showed bilateral polycystic ovaries.

CASE III (PCOD): A 26 year old lady, married 11 years with PCOD had ovarian drilling done. Her hormonal profiles were FSH=3.55 mIU/ml and LH=11.05 mIU/ml and Prolactin=11.05Ng/ml. Ultrasonography confirmed bilateral polycystic ovaries. All women were anovulatory. Polycystic ovaries have characteristic necklaces of 10 or more primordial or primary follicles (2-10mm) but have few mature follicles. Most women are anovulatory. Polycystic ovaries are also found in normal women who are treated by IVF. These patients are now used to retrieve follicles and oocytes for in vitro maturation (IVM) , without ovarian stimulation (Trounson et al, 1988)

POLYCYSTIC OVARY (26 YEARS)

Cortex

Follicles

Medulla

Epithelium

Hilum

The cortex shows a necklace of primordial & primary follicles, characteristic of these ovaries. Note medulla and hilum. x50

POLYCYSTIC OVARY– PRIMORDIAL FOLLICLES Medulla

Blood vessels Cortex Follicles

Epithelium

Three primordial follicles are seen in the cortex and the medulla has blood vessels. x100

POLYCYSTIC OVARY (26 YEARS) –EPITHELIUM

Light cells

Cortex

Epithelium Dark cells

The epithelium shows light and dark cells – cuboidal to lower columnar. x1000

POLYCYSTIC OVARY – PRIMORDIAL FOLLICLES Stroma

Follicles

Cortex

A nest of primordial is seen in the ovarian cortex. Such ovaries are used to obtain follicles for oocyte maturation in vitro. x400

POLYCYSTIC OVARY – PRIMORDIAL FOLLICLE Stroma

Follicle cells

Oocyte Theca

Cortex

Both light and dark cells form the wall of the follicle x1000

COVER PICTURE: PRIMORDIAL FOLLICLE-PCOD (TEM)

Stroma Nucleolus

Follicle Cells

Nucleus

Mitochondria

Balbiani Body

The diplotene oocyte at prophase has a single layer of follicle cells surrounded by stroma. Note large nucleus and Balbiani body x 1000

PRIMORDIAL OOCYTE (PCOD) – BALBIANI BODY Annulate lamellae

Golgi

Balbiani body

Mitochondria Nucleus

Lipofuschin

Balbiani body consists predominantly of mitochondria,lipofuschin, Golgi, granular material and annulate lamellae x5000

PRIMORDIAL FOLLICLE (PCOD) Basal lamina

Stroma

Mitochondria

Tunnel Follicle cell

Microvilli on oocyte surface are pressed against follicle cells. Note tunnels that contained follicle cell processes within oocyte. x3000

EARLY GROWING FOLLICLE (PCOD) – ZONA SECRETION Follicle cell

Follicle cell

Annulate lamellae

Pockets of secreted zona material are seen between follicle cells and within oocyte(arrows) , probably secreted by both. x6000

GROWING FOLLICLE (PCOD) WITH SECRETED ZONA Basal lamina

Dead cell

Follicle cells

Oocyte

Theca

Follicle is multilayered and the zona (gold) has been secreted around oocyte. Note basal lamina. x800

GROWING FOLLICLE (PCOD) – ZONA SECRETION Follicle cells

Basal lamina

Oocyte

Mitochondria Nucleus Nucleolus

The follicle has 2 layers of cells and the zona is secreted in pockets (arrows) outside oocyte. Note thickened basal lamina. x2000

GROWING FOLLICLE (PCOD) – CORONA CELL PROCESSES (GAP JUNCTIONS)

Follicle cells

Microvilli

Zona

Oocyte

Mitochondria

The zona is thick and oocyte microvilli penetrate the inner zona. Note follicle cell processes in ooplasm (arrowheads) . x6000

CORPUS LUTEUM (PCOD) Blood vessel Theca interna

Cavity

Granulosa Lutein cells

Primordial follicle

The corpus luteum is formed after ovulation. The granulosa cells are luteinized and secrete progesterone. x200

CORPUS LUTEAL CELLS (PCOD)

Capillary

Lutein cells

The granulosa lutein cells have prominent and lipid-rich cytoplasm. x1000

PRIMORIAL FOLLICLE (PCOD) – EARLY ATRESIA? Follicle cell

Lipofuscin

Ooplasm

Lipofuscin

Nucleus Mitochondria

The ooplasm contains many lipofuscin bodies(ageing pigment) associated with the Balbiani body. x5000

GOLGI COMPLEX (PCOD) – PRIMARY FOLLICLE

RER

Nucleus

Ooplasm

Golgi consists of flattened sacs (cisternae) and rounded vesicles embedded in a dense matrix. x20,000

OOCYTE NUCLEOLUS (PCOD) – PRIMARY FOLLICLE Dense body

Nucleus

The nucleolus is highly reticulated and associated with a dense body. x15,000

BLOOD CAPILLARY - OVARIAN CORTEX (PCOD) Collagen microfibrils

Pericyte

Stroma

RBC Endothelium

The capillary has a lining of endothelial cells and contains red blood corpuscles(RBC) . Capillaries also associate with follicles. x3000

POF OVARIES – PATIENTS (N=3) Premature Ovarian Failure (POF) represents a condition in which the ovaries have undergone differentiation and have functioned transiently from puberty but cease to function prior to 40 years. It affects about 1% of the general population (Vegetti et al.1998). Some patients with premature ovarian failure have normal appearing follicles in the ovary that inexplicably fail to function despite high gonadotropin levels The incidence of POF is 1 in 100 by the age of 40 and 1 in 1000 by the age of 30. It contributes to 10-28 % of primary amenorrhoea. During a 2-year study, we came across 180 patients with (POF), out of a total of 3600 patients attending the Fertility Clinic. Of these, 100 patients were taken for our study. In summary:-The incidence of POF was 5 %. 24 patients attained menarche between 10-12 years of age, 50 patients between 13-15 years and 26 patients between 16-18 years. 80% of the patients had menstrual cycles on induction and 20 % had irregular cycles (Selvaraj Ph.D. thesis) Its main causes include:1) Ovarian follicle depletion - A mechanism of ovarian failure caused by accelerated follicular atresia or deficient follicle number. 2) Ovarian follicle dysfunction - A mechanism of ovarian failure caused by failure of normal appearing follicles to function normally in spite of high gonadotropin levels. The pregnancy outcome was 31.9% in patients with POF in our oocyte donation program in Chennai, when compared to 22.3% in the perimenopausal group. This was largely due to hormonal replacement therapy creating a better environment for the implantation of the embryo. Our TEM study of 3 POF patients show no evidence of functional follicles. There were a few atretic follicles in the cortex, large bundles of smooth muscle in medulla and abnormal blood vasculature.

OVARY – PREMATURE OVARIAN FAILURE (POF) Epithelium

Cortex Blood capillary

Medulla

Smooth muscle

Hilum

The ovary has no follicles in cortex. Note blood capillaries and large bundle of smooth muscle in medulla. x100

EPITHELIUM & CORTEX - OVARY(POF) Epithelium

Helicine blood vessel Tunica albuginea Cortex

Coelomic epithelium is a single layer of cells outside the tunica albuginea(dense connective tissue) . No follicles in cortex. x400

EPITHELIUM – OVARY (POF)

Epithelia cells

Tunica albuginea Cortex

The coelomic epithelium shows a single layer of cuboidal cells. x2000

PRIMORDIAL FOLLICLE OOCYTE (POF) - ATRESIA Follicle cell

Theca Oocyte

Nucleus Balbiani body

Nucleolus

Vacuoles

Follicles are rare and are atretic. Note degenerating cytoplasm with vacuoles and disorganized follicle cells. x2000

BLOOD CAPILLARY IN CORTEX (POF)

RBC Endothelium Stroma

Pericyte

Note disorganized endothelium and pericytes. The stroma is translucent. x3000

BUNDLE OF SMOOTH MUSCLE – OVARY(POF) Stroma

Smooth muscle cells

Smooth muscle is commonly seen in POF ovaries. The cells have prominent nuclei and dense bodies (arrowheads) . x2500

RARE CORPUS LUTEUM & CAPILLARY (POF) Epithelium

Corpus luteum

Capillary

The corpus luteum which consists of granulosa luteal cells associated with blood capillaries. x200

PREOVULATORY OOCYTE MATURATION IN VITRO Preovulatory oocytes are those retrieved for IVF and ICSI, just prior to ovulation. The final stages of oocyte maturation are usually completed in vitro after induction of ovarian stimulation with gonadotrophins. All these stages from germinal vesicle (GV) oocyte to mature oocyte (Metaphase II) have been well documented (Sathananthan, 1985, 1997; Sathananthan et al,1993) and will be dealt with very briefly to complete oogenesis. At oocyte retrieval, some oocytes are still immature and are at stages ranging from metaphase I (MI) to metaphase II (MII). However, with recent ovarian stimulation protocols using GNRH agonists/FSH and better timing of HCG, the majority of oocytes approach MII and could be easily harvested for ART and identified, particularly when intracytoplasmic sperm injection (ICSI) is the procedure of choice. This technique involves partial removal of cumulus cells, which enables visualization of the first polar body (PB1). Hence the preincubation time of oocytes before IVF is minimized and sperm injection could be done when the oocyte is just ripe and not pre-mature or post-mature. This is how it occurs in nature at ovulation. However, growing oocytes and germinal vesicle (GV) oocytes and even primordial follicles may be aspirated during oocyte retrieval, which need to be cultured in vitro to full maturity before they are used for either, IVF or ICSI (Trounson, et al. 1998). Germinal vesicle breakdown is a critical stage when follicle cells retract from the oocyte –surface, uncoupling cell junctions. The second maturation division is completed only at fertilization, when the sperm fuses with the mature oocyte and introduces the centrosome.

EARLY GRAAFIAN FOLLICLE - GROWING OOCYTE Zona Cortical granules

Golgi

Microvilli

Nucleolus RER

Nucleus

Oocyte Mitochondria

Oocyte has a reticulated nucleolus, RER, Golgi, cortical granules and mitochondria. Note microvilli on surface.X3500 (Backcover)

GERMINAL VESICLE BREAK-DOWN (GVBD) AT PREOVULATORY MATURATION

Zona

GV

Sperm Cumulus Chromosomes

Vacuoles

Egg Sperm Zona

GV oocyte

GVBD

Germinal vesicle (GV) egg has an eccentric nucleus and is atretic. The GV has broken down at the onset of meiotic maturation (right). Note uncoupling of cumulus cells at GVBD. x1000,x400

CORONA CELL PROCESS IN ZONA - GV OOCYTE Corona cell

Zona Process

The process penetrates the zona and extends to the surface of the egg. Note microfilaments in process. x 35,700 (Sathananthan 1985)

GERMINAL VESICLE OOCYTE

Oocyte has 1or2 layers of cortical granules and tunnels that had follicular cell processes. Note dense compact nucleolus x2500

CORONA CELL PROCESSES IN GV EGG Zona

Ooplasm

Microvilli

Gap junction

Tunnels

Cortical granule

Mitochondria Lysosomes

Corona cell processes (green) are found in tunnels and form gap junctions within ooplasm. x20,500 (Sathananthan, 1997)

MATURE OOCYTES WITH CUMULUS (IVF)

The cumulus cells are retracting from the surface of each egg via the zona. The eggs were at MI & MII of maturation. x100

MATURE METAPHASE II EGG AT OVULATION

Polar body

Cumulus

Oocyte has one polar body and chromosomes in ooplasm (arrow) . Cumulus has retracted from zona x400 (Sathananthan, 1985)

MATURE METAPHASE II OOCYTE - SPINDLE Zona Cortical granules

Chromosomes

Spindle

Mitochondria

Note barrel-shaped spindle with chromosomes at equator.Maternal centrosome is non-functional. x9100 (Sathananthan et al, 1988)

TELOPHASE I EGG – 1st POLAR BODY ABSTRICTION

Note chromosomes in polar body and in oocyte. The mid-body is the point of abstriction. x6000 (Sathananthan, 2000)

TELOPHASE II EGG – 2nd POLAR BODY ABSTRICTION

Second polar body is abstricted at fertilization. Note chromosomes in polar body and ooplasm. x7000 (Sathananthan,1985)

ULTRASTRUCTURE OF HUMAN OOCYTE

Composite diagram of cytoplasmic components of immature & mature eggs. Note corona cell junction. (Sathananthan et al 1993)

RELEVANT REFERENCES De Pol, A., Vaccina, F., Forabosco, A., et al. (1997) Apoptosis of germ cells during human prenatal oogenesis, Hum. Reprod. 12: 2235 – 2241. Franks, S. (1995) Polycystic ovary syndrome. N.Eng.J.Med. 333:853-861 Gougeon, A. (1997) Kinetics of human ovarian follicular development. In: Microscopy of Reproduction and Development: A Dynamic Approach, P.M. Motta, ed. Antonio Delfino Editore, Rome, pp. 67 – 77. Hartshorne, G.M. (1997) In vitro culture of ovarian follicles. Rev. Reprod. 2: 94 – 104. Hovatta, O., Wright, C., Krausz, T., et al (1999) Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation., Hum.Reprod., 14: 2519 - 2524. Motta, P.M., ed (1997) Microscopy of Reproduction and Development: a Dynamic Approach. Antonio Defino Editore. Rome. Motta, P.M., Makabe, S., Naguro, T., et al. (1994) Oocyte – follicle cells association during development of human ovarian follicle: A study by high-resolution scanning and transmission electron microscopy. Arch. Histol. Cytol., 57: 369 – 394. Motta, P.M., Nottola, S.A, Familiari, G., et al. (1995) Ultrastructure of human reproduction from folliculogenesis to early embryo development. A review. It. J. Anat. Embryol., 100: 9 – 52. Motta, P.M, Nottola, S.A, Makabe, S., et al (2000) Mitochondrial morphology in human fetal and adult female germ cells. Human Reprod.,15(Suppl.2) 129-147. Sathananthan, A.H. (1985) Maturation of the human oocyte in vitro: Nuclear events during meiosis (an ultrastructural study). Gamete Res. 12: 237 – 254. Sathananthan AH et al (2006). From oogonia to mature oocytes: inactivation of the maternal centrosome in humans,. Micros Res Tech 69: 396-407 (Review)

RELEVANT REFERENCES Sathananthan, A.H., Trounson, A.O., Freeman, L., et al. (1988) The effects of cooling human oocytes. Hum. Reprod., 3: 968 – 977. Sathananthan, A.H. (1997) Ultrastructure of the human egg. Hum. Cell., 10: 21 – 38. Sathananthan, A.H., Ng, S.C., Bongso, A., et al. (1993) Visual Atlas of Early Human Development for Assisted Reproductive Technology. Serono, Singapore, pp. 209. Sathananthan, A.H., Selvaraj, K., and Trounson, A.O. (2000) Fine structure of human oogonia in the foetal ovary. Mol. Cell. Endocrin., 161: 3 – 8. Sathananthan, A.H., Selvaraj, K. Lakshmi, M.S. et al (2002) From oogonia to mature oocytes: Inactivation of the maternal centrosomes in humans. (in press) Shaw, J.M., Dawson, K.J., and Trounson, A.O. (1997) A critical evaluation of ovarian tissue cryopreservation and grafting as a strategy for preservation of the human female germline. Reprod. Med. Rev., 6: 163 – 183.

Trounson, A., Anderiesz, C., Jones, G.M. et al (1998) Oocyte maturation. Hum. Reprod. 13: (Suppl. 3) 52 – 62. Trounson AO, Gosden RG (eds) (2003) Biology and Pathology of the Oocyte. Cambridge University Press. pp 350. Veeck, L.L. (1999) An Atlas of Human Gametes and Conceptuses: An illustrated reference for assisted reproductive technology, Parthenon Publishing, New York. Vegetti,W.,Tibiletti,M.G.,Testa,G. et al (1998) Inheritance in idiopathic premature ovarian failure:analysis of 71 cases.Hum.Reprod.13:1796-1800. Wartenberg, H., (1989) Ultrastructure of foetal ovary including oogenesis. In: Ultrastructure of Human Gametogenesis and Early Embryogenesis, J. Van Blerkom and P.M. Motta, eds. Kluwer Academic, Boston, pp. 61 – 84.

ACKNOWLEDGEMENTS We thank the Monash Institute of Reproduction and Development (MIRD) and GG Hospital , Chennai, for the research facilities for the production of this CDROM. Dr A.H.S. is particularly indebted to the Directors of MIRD, Prof. David de Kretser, Prof. Alan Trounson and Prof. Richard Boyd for their continued encouragement, support and direction. Henry has completed 37 years of research at Monash University in an honorary capacity. Special thanks are due to Prof. R.G. Edwards (Cambridge) who inspired Henry to publish on-line, as CD –ROM editor of Human Reproduction Update (1995-2000). We also thank the Late Prof. S .S. Ratnam (Singapore) for his encouragement and direction, and who was particularly helpful to Sri Lanka, India, Pakistan & South East Asia. Finally, Henry thanks Dr K. Selvaraj (Chennai), Dr S. Gunasheela (Bangalore) and Dr V. Metgud (Belgaum) for giving him the opportunity to collaborate in research and his numerous friends and well wishers who have invited him for conferences and workshops in India (since 1985 ) and above all for their hospitality. We thank the authors and publishers for permission to reproduce some images in this CD

THE AUTHORS AND CLINICIANS

Dr K.Selvaraj Dr.A.H.Sathananthan Dr.P.Selvaraj Dr.V.Ganesh Dr.M.S.Lakshmi