Fundamentals of reproduction

This topic contains Gametogenesis- oogenesis and spermatogenesis, ovulation, fertilization, development of fertilized ovum/ zygote, implantation, development of decidua, chorion and chorionic villi, development of inner cell mass.

1. FUNDAMENTALS OF REPRODUCTION BY, MS. PRIYANKA GOHIL MSc (N) OBG PhD SCHOLAR

2. CONCEPTION GAMETOGENESIS The process involved in the maturation of the two highly specialized cells, spermatozoon in male and ovum in female before they unite to form zygote, is called gametogenesis.

3. OOGENESIS The process involved in the development of a mature ovum is called oogenesis. The primitive germ cells take their origin from the yolk sac at about the end of 3 rd week and their migration to the developing gonadal ridge is completed round about the end of 4 th week. In the female gonads, the germ cells undergo a number of rapid mitotic divisions and differentiate into oogonia. The number of oogonia reaches its maximum at 20th week, numbering about 7 million.

4. While the majority of the oogonia continue to divide, some enter into the prophase of the first meiotic division and are called primary oocytes. These are surrounded by flat cells and are called primordial follicles and are present in the cortex of the ovary. At birth, there is no more mitotic division and all the oogonia are replaced by primary oocytes which have finished the prophase of the first meiotic division and remain in resting phase (dictyotene stage) between prophase and metaphase. Total number of primary oocyte s at birth is estimated to be about 2 million.

5. The primary oocytes do not finish the first meiotic division until puberty is reached. At puberty, some 400,000 primary oocytes are left behind, the rest being atretic. Out of these, some 400 are likely to ovulate during the entire reproductive period.

6. Maturation of the oocytes :- The essence of maturation is reduction of the number of chromosomes to half. Before the onset of first meiotic division, the primary oocytes double its DNA by replication, so they contain double the amount of normal protein content. There are 22 pairs of autosomes which determine the body characteristics and 1 pair of sex chromosomes, named “XX”. The first stage of maturation occurs with full maturation of the ovarian follicle just prior to ovulation but the final maturation occurs only after fertilization.

7. The primary oocyte undergoes first meiotic division giving rise to secondary oocyte and one polar body. The two are of unequal size, the secondary oocyte contains haploid number of chromosomes (23, X), but nearly all the cytoplasm and the small polar body also contains half of the chromosomes (23, X) but with scanty cytoplasm. Ovulation occurs soon after the formation of the secondary oocyte.

8. The secondary oocyte completes the second meiotic division (homotypical) only after fertilization by the sperm in the Fallopian tube and results in the formation of two unequal daughter cells, each possessing 23 chromosomes (23, X), the larger one is called the mature ovum and the smaller one is the second polar body containing the same number of chromosomes. The first polar body may also undergo the second meiotic division. In the absence of fertilization, the secondary oocyte does not complete the second meiotic division and degenerates as such.

9. Chromosome nomenclature :- The number designates the total number of chromosomes (in numerals) followed by the sex chromosome constitution after the comma.

10. Structure of a mature ovum :- A fully mature ovum is the largest cell in the body and is about 130 microns in diameter. It consists of cytoplasm and a nucleus with its nucleolus which is eccentric in position and contains 23 chromosomes (23, X). During fertilization, the nucleus is converted into a female pronucleus. The ovum is surrounded by a cell membrane called vitelline membrane. There is an outer transparent mucoprotein envelope, the zona pellucida.

11. The zona pellucida is penetrated by tiny channels which are thought to be important for the transport of the materials from the granulosa cells to the oocyte. In between the vitelline membrane and the zona pellucida, there is a narrow space called perivitelline space which accommodates the polar bodies. The human oocyte, after its escape from the follicle, retains a covering of granulosa cells known as the corona radiata derived from the cumulus oophorus.

12. SPERMATOGENESIS The process involved in the development of spermatids from the primordial male germ cells and their differentiation into spermatozoa is called spermatogenesis. Shortly before puberty, the primordial germ cells develop into spermatogonia and remain in the wall of seminiferous tubules. The spermatogonia, in turn, differentiate into primary spermatocytes which remain in the stage of prophase of the first meiotic division for a long time (about 16 days). Each spermatocyte contains 22 pairs of autosomes and 1 pair of sex chromosomes, named “XY”.

13. With the completion of the first meiotic division, two secondary spermatocytes are formed having equal share of cytoplasm and haploid number of chromosomes either 23, X or 23, Y. Then immediately follows the second meiotic division (homotypical) with the formation of four spermatids, each containing haploid number of chromosomes, two with 23, X and two with 23, Y. Immediately after their formation, extensive morphological differentiation of the spermatids occurs without further cell division to convert them into spermatozoa. The process is called spermiogenesis. In man, the time required for a spermatogonium to develop into a mature spermatozoon is about 61 days.

14. Sperm capacitation and acrosome reaction : - Capacitation is the physiochemical change in the sperm by which it becomes hypermotile and is able to bind and fertilize a secondary oocyte. Capacitation takes place in the genital tract and takes about 2–6 hours. The changes involve cyclic AMP dependent phosphorylation with increase in intracellular pH (influx of Ca ++ and efflux of H + ). Activation of acrosomal membranes causes release of hyaluronidase, hydrolytic enzymes, proacrosin, acrosin, that help the sperm to digest the zona pellucida and to enter into the oocyte. During acrosomal reaction the sperm plasma membrane fuses with the outer acrosomal membrane .

15. The sperm with acrosomal membrane bind the Zona Protein (ZP3 ), after passing between the corona radiata cells. After acrosome reaction, the sperm binds to Zona Protein ZP2. Then there is zona reaction to prevent polyspermy. Acrosome sperm penetrate the zona pellucida → reaches the perivitelline space → fuses with the oocyte plasma membrane. The sperm head swells and the fused membrane vesiculates. The whole sperm head, midpiece and tail are drawn into the cytoplasm. Gamete fusion is an integrin mediated process. About 3–6 hours after insemination, one polar and two pronuclear bodies are visible and they migrate to the center of the oocyte.

16. Fertilization in vitro : - Capacitation and acrosome reaction occur within few hours in simple media. Washed and motile sperm (2 × 105/mL) are added to the oocyte. In ICSI (Intracytoplasmic sperm injection), microinjection of a single sperm into the oocyte is done.

17. Structure of a mature spermatozoon :- It has got two parts, a head and a tail. The head consists principally of the condensed nucleus and acrosomal cap. Acrosome is rich in enzymes. The tail is divided into four zones — the neck, the middle piece, the principal piece and the end piece.

18. OVULATION Ovulation is a process whereby a secondary oocyte is released from the ovary following rupture of a mature Graafian follicle and becomes available for conception. Only one secondary oocyte is likely to rupture in each ovarian cycle which starts at puberty and ends in menopause. In relation to the menstrual period, the event occurs about 14 days prior to the expected period. However, menstruation can occur without ovulation and ovulation remains suspended during pregnancy and lactation.

19. MECHANISM : - The process of ovulation is a complex one. Preovulatory changes occur both in the follicle and the oocyte. Changes in the follicle : - There is preovulatory enlargement of the Graafian follicle due to accumulation of follicular fluid and measures about 20 mm in diameter . The cumulus oophorus separates from the rest of the granulosa cells and floats freely in the antrum.

20. The inner layer of the cells surrounding the oocyte is arranged radially and is termed corona radiata. The follicular wall near the ovarian surface becomes thinner. The stigma develops as a conical projection which penetrates the outer surface layer of the ovary and persists for a while (½ – 2 minutes) as a thin membrane. The cumulus escapes out of the follicle as a slow oozing process, taking about 1–2 minutes along with varying amount of follicular fluid. The stigma is soon closed by a plug of plasma.

21. Changes in the oocyte :- Significant changes in the oocyte occur just prior to ovulation (few hours). Cytoplasmic volume is increased along with changes in the number, distribution of mitochondria and in the Golgi apparatus. Completion of the arrested first meiotic division occurs with extrusion of first polar body, each containing haploid number of chromosomes (23, X).

22. CAUSES :- The following are the possible explanations which may operate singly or in combination: Endocrinal LH surge: Sustained peak level of estrogen for 24–36 hours in the late follicular phase → LH surge occurs from the anterior pituitary. Ovulation approximately occurs 16–24 hours after the LH surge. LH peak persists for about 24 hours. The LH surge stimulates completion of reduction division of the oocyte and initiates luteinization of the granulosa cells, synthesis of progesterone and prostaglandins.

23. FSH rise: Preovulatory rise of progesterone facilitates the positive feedback action of estrogen to induce FSH surge → increase in plasminogen activator → plasminogen → plasmin → helps lysis of the wall of the follicle. Thus, the combined LH/FSH midcycle surge is responsible for the final stage of maturation, rupture of the follicle and expulsion of the oocyte.

24. Stretching factor: It is more a passive stretching than rise in intrafollicular pressure which remains static at about 15 mm Hg. Contraction of the micromuscles in the theca externa and ovarian stroma due to increased prostaglandin secretion.

25. EFFECT OF OVULATION :- Following ovulation, the follicle is changed into corpus luteum. The ovum is picked up into the Fallopian tube and undergoes either degeneration or further maturation, if fertilization is to occur. Menstruation is unrelated with ovulation and anovular menstruation is quite common during adolescence, following childbirth and in women approaching menopause.

26. FERTILIZATION Fertilization is the process of fusion of the spermatozoon with the mature ovum. It begins with sperm egg collision and ends with production of a mononucleated single cell called the zygote. Its objectives are: To initiate the embryonic development of the egg To restore the chromosome number of the species. Almost always, fertilization occurs in the ampullary part of the uterine tube.

27. APPROXIMATION OF THE GAMETES : - The ovum, immediately following ovulation is picked up by the tubal fimbriae which partly envelope the ovary, especially at the time of ovulation. The pick up action might be muscular or by a kind of suction or by ciliary action or by a positive chemotaxis exerted by the tubal secretion. The ovum is rapidly transported to the ampullary part. Fertilizable life span of oocyte ranges from 12 to 24 hours whereas that of sperm is 48 to 72 hours.

28. Out of hundreds of millions of sperms deposited in the vagina at single ejaculation, only thousands capacitated spermatozoa enter the uterine tube while only 300–500 reach the ovum. The tubal transport is facilitated by muscular contraction and aspiration action of the uterine tube. It takes only few minutes for the sperm to reach the Fallopian tube.

29. CONTACT AND FUSION OF THE GAMETES: - Complete dissolution of the cells of the corona radiata occurs by the chemical action of the hyaluronidase liberated from the acrosomal cap of the hundreds of sperm present at the site . Penetration of the zona pellucida is facilitated by the release of hyaluronidase from the acrosomal cap. More than one sperm may penetrate the zona pellucida. Out of the many sperms, one touches the oolemma. Soon after the sperm fusion, penetration of other sperm is prevented by zona reaction (hardening) and oolemma block. cortical granules by exocytosis from the oocyte.

30. Completion of the second meiotic division of the oocyte immediately follows, each containing haploid number of chromosomes (23, X). The bigger one is called the female pronucleus and the smaller one is called second polar body which is pushed to the perivitelline space. In the human, both the head and tail of the spermatozoon enter the cytoplasm of the oocyte but the plasma membrane is left behind on the oocyte surface. Head and the neck of the spermatozoon become male pronucleus containing haploid number of chromosomes (23, X) or (23, Y).

31. The male and the female pronuclei unite at the center with restoration of the diploid number of chromosomes (46) which is constant for the species. The zygote, thus formed, contains both the paternal and maternal genetic materials. In some instances, an antigen called fertilizin present on the cortex and its coat of the ovum, reacts with the antibody called antifertilizin liberated at the plasma membrane of the sperm head. Thus the union between the two gametes may be an immunological reaction (chemotaxis).

32. Sex of the child is determined by the pattern of the sex chromosome supplied by the spermatozoon. If the spermatozoon contains ‘X’ chromosome, a female embryo (46, XX) is formed; if it contains a ‘Y’ chromosome, a male embryo (46, XY) is formed.

33. DEVELOPMENT OF FERTILIZED OVUM / ZYGOTE

34. MORULA After the zygote formation, typical mitotic division of the nucleus occurs by producing two blastomeres. The two cell stage is reached approximately 30 hours after fertilization. Each contains equal cytoplasmic volume and chromosome numbers. The blastomeres continue to divide by binary division through 4, 8, 16 cell stage until a cluster of cells is formed and is called morula, resembling a mulberry.

35. As the total volume of the cell mass is not increased and the zona pellucida remains intact, the morula after spending about 3 days in the uterine tube enters the uterine cavity through the narrow uterine ostium (1 mm) on the 4th day in the 16-64 cell stage. The transport is a slow process and is controlled by muscular contraction and movement of the cilia. The central cell of the morula is known as inner cell mass which forms the embryo proper and the peripheral cells are called outer cell mass which will form protective and nutritive membranes of the embryo .

36. BLASTOCYST While the morula remains free in the uterine cavity on the 4 th and 5 th day, it is covered by a film of mucus. The fluid passes through the canaliculi of the zona pellucida which separates the cells of the morula and is now termed blastocyst. Zona hatching is the next step so that trophectoderm cells interact with endometrial cells and implantation occurs. Due to blastocyst enlargement the zona pellucida becomes stretched, thinned and gradually disappears. Lysis of zona and escape of embryo is called zona hatching.

37. The cells on the outer side of the morula (polar) become trophectoderm and the inner cells (apolar) become inner cell mass by the mediation of epithelial cadherin (E-cadherin) (protein). Trophectoderm differentiates into chorion(placenta) and the inner cell mass into the embryo. Completely undifferentiated cells are called the pluripotent embryonic stem (ES) cells. E mbryonic stem (ES) cells are able to produce mature somatic cells of any germ layer (ectoderm, mesoderm and endoderm).

38. TROPHOBLAST As, the cells of the blastocyst differentiate into an outer trophectoderm and an inner cell mass. Just before implantation, the trophectoderm is further differentiated into an inner mononuclear cellular layer called cytotrophoblast or Langhan's layer and an outer layer of multinucleated syncytium called syncytiotrophoblast. The cytotrophoblasts that line the villous stems are the villous cytotrophoblasts. The cytotrophoblast cells that invade the decidua are known as ‘interstitial extravillous cytotrophoblast’ and those that invade the lumens of the maternal spiral arteries are known as ‘intravascular extravillous cytotrophoblast’.

39. Throughout pregnancy, syncytiotrophoblast is derived from the cytotrophoblast. Placenta and the fetal membranes are developed from the trophoblast. It is involved in most of the functions ascribed to the placenta as a whole. Thus, it serves at least 3 important functions — invasion, nutrition and production of hormones for the maintenance of pregnancy. Local cytokines regulate the invasion of the cytotrophoblasts in the decidua.

40. IMPLANTATION (Syn: Nidation) Implantation occurs in the endometrium of the anterior or posterior wall of the body near the fundus on the 6 th day which corresponds to the 20 th day of a regular menstrual cycle. Implantation occurs through four stages e.g. apposition, adhesion, penetration and invasion.

41. CHANGES IN THE BLASTOCYST: The polar trophoblast cells adjacent to the inner cell mass are primarily involved in adhesion to the endometrial cells. The factors responsible for blastocyst attachment are: P. selectin, heparin sulfate, proteoglycan, EGF (Epidermal growth factor) , integrins, trophinin and others. The signals for trophoblast multiplication arise from the inner cell mass.

42. ENDOMETRIUM AT THE IMPLANTATION SITE : The endometrium is in the secretory phase corresponding to 20–21 days of cycle. The microvilli on the surface of the trophectoderm interdigitate with the decidual cells to form the junctional complexes. Endometrial receptivity and molecular signaling during implantation is induced by progesterone, LIF (leukemia inhibitory factor), prostaglandins and COX-2 (Cyclooxygenase-2) .

43. APPOSITION: Occurs through pinopod formation. Pinopods are long finger like projections (microvilli) from the endometrial cell surface. These pinopods absorb the endometrial fluid which is secreted by the endometrial gland cells. This fluid, rich in glycogen and mucin provides nutrition to the blastocyst initially. Unless this fluid is absorbed, adhesion phase cannot occur. Adhesion of blastocyst to the endometrium occurs through the adhesion molecules like integrin, selectin and cadherin (glycoproteins).

44. PENETRATION: Actual penetration and invasion occur through the stromal cells in between the glands and is facilitated by the histolytic action of the blastocyst. With increasing lysis of the stromal cells, the blastocyst is burrowed more and more inside the stratum compactum of the decidua. Vacuoles appear in the advancing syncytium which fuse to form large lacunae. These are more evident at the embryonic pole. Concurrently, the syncytial cells penetrate deeper into the stroma and erode the endothelium of the maternal capillaries.

45. The syncytium by penetrating the vessels, not only becomes continuous with the endothelial lining but permits the maternal blood to enter into the lacunar system. Ultimately erosion of few maternal arteries with formation of blood space (lacunae) occurs. Nutrition is now obtained by aerobic metabolic pathway from the maternal blood. Further penetration is stopped probably by the maternal immunological factor and the original point of entry is sealed by fibrin clot and later by epithelium. The process is completed by 10th or 11th day which corresponds to D 24-25 from LMP

46. This type of deeper penetration of the human blastocyst is called interstitial implantation and the blastocyst is covered on all sides by the endometrium (decidua). Occasionally, there may be increased blood flow into the lacunar spaces at the abembryonic pole. This results in disruption of the lacunae and extravasation of blood into the endometrial cavity. This corresponds approximately to 13th day after fertilization (at about the expected day of the following period). This may produce confusion in determination of the expected date of delivery.

47. The process of implantation is controlled by the immuno-modulatory role of various cytokines (interleukins 3, 4, 5, 6, 10, 13), many local peptides like epidermal growth factor (EGF), insulin like growth factor (IGF) and prostaglandins. Both the decidua and the embryo synthesize these molecules.

48. THE DECIDUA The decidua is the endometrium of the pregnant uterus. It is so named because much of it is shed following delivery. Decidual reaction: The increased structural and secretory activity of the endometrium that is brought about in response to progesterone following implantation is known as decidual reaction .

49. Changes occur in all the components of the endometrium but most marked at the implantation site and first commence around maternal blood vessels. The fibrous connective tissues of the stroma become changed into epithelioid cells called decidual cells. The glands show marked dilatation and increased tortuosity with its lining epithelium showing evidences of active cell proliferation with increased secretory activity. There are areas of small interstitial hemorrhage and leukocytic infiltration specially at the implantation site.

50. The well developed decidua differentiates into three layers: Superficial compact layer consists of compact mass of decidual cells, gland ducts and dilated capillaries. The greater part of the surface epithelium is either thinned out or lost. Intermediate spongy layer (cavernous layer) contains dilated uterine glands, decidual cells and blood vessels. It is through this layer that the cleavage of placental separation occurs. Thin basal layer containing the basal portion of the glands and is opposed to the uterine muscle. Regeneration of the mucous coat occurs from this layer following parturition.

51. After the interstitial implantation of the blastocyst into the compact layer of the decidua, the different portions of the decidua are renamed as:- Decidua basalis or serotina: the portion of the decidua in contact with the base of the blastocyst Decidua capsularis or reflexa : the thin superficial compact layer covering the blastocyst and Decidua vera or parietalis : the rest of the decidua lining the uterine cavity outside the site of implantation. Its thickness progressively increases to maximum of 5–10 mm at the end of the second month and thereafter regression occurs with advancing pregnancy so that beyond 20th week, it measures not more than 1 mm.

52. As the growing ovum bulges towards the uterine cavity, the space between the decidua capsularis and the decidua vera, called the decidual space is gradually narrowed down and by 4th month, it is completely obliterated by the fusion of the decidua capsularis with the decidua vera. At term, they become atrophied due to pressure and the two cannot be defined as a double layer. The decidua basalis, however, retains its characteristic appearance till term and becomes the maternal portion of the placenta.

53. Functions: It provides a good nidus for the implantation of the blastocyst. It supplies nutrition to the early stage of the growing ovum by its rich sources of glycogen and fat. Deeper penetration of the trophoblast is controlled by local peptides, cytokines and integrins. Decidua basalis takes part in the formation of basal plate of the placenta.

54. CHORION AND CHORIONIC VILLI The chorion is the outermost layer of the two fetal membranes (chorion and amnion). It consists of two embryonic layers :- outer trophoblast and inner primitive mesenchyme which appears on 9th day. At the beginning of the 3rd week, the syncytiotrophoblast produces irregular finger like projections which are lined internally by the cytotrophoblast. These finger like buds are called primary stem villi:- surrounded by lacunar spaces which will later form into intervillous spaces.

55. After the appearance of the primitive mesenchyme and the development of the chorion, the primary stem villi are named chorionic villi. With the insinuation of the primary mesoderm into the central core of the villi structures, secondary villi are formed on 16th day. Later on mesodermal cells in the villi begin to differentiate into blood cells and blood vessels, thus forming villous capillary system. These vascularized villi are called tertiary villi which are completed on 21st day. Later on, this extra embryonic circulatory system establishes connection with the intraembryonic circulatory system through the body stalk.

56. Meanwhile, the cytotrophoblastic cells beyond the tips of the villus system penetrate into the overlying syncytium adjacent to the decidua. The cells become continuous with those of the neighboring villus system traversing through the syncytium. Thus, a thin outer cytotrophoblastic shell is formed which surrounds the entire blastocyst. The zone of the decidua immediately adjacent to the trophoblastic shell is called trophosphere which comprises of the compact layer of the decidua. Fibrinoid deposit appears on the syncytiotrophoblast outside the trophoblastic shell andis called Nitabuch’s membrane.Maternal blood vessels pass through all the layers to reach the intervillous space.

57. The villi overlying the decidua basalis continue to grow and expand and are called chorion frondosum which subsequently forms the discoid placenta. The chorionic villi on the decidua capsularis gradually undergoes atrophy from pressure and become converted into chorion laeve by the 3rd month and lies intervening between the amnion and decidua on its outer surface. Remnant of decidual cells and of the trophoblast can however be distinguished microscopically.

58. DEVELOPMENT OF INNER CELL MASS Along with the changes in the trophoblast, on the 8th day, the embryoblast differentiates into bilaminar germ disc which consists of dorsal ectodermal layer of tall columnar cells and ventral endodermal layer of flattened polyhedral cells. The bilaminar germ disc is connected with the trophoblast by mesenchymal condensation, called connecting stalk or body stalk which later on forms the umbilical cord.

59. Two cavities appear one on each side of the germ disc. On 12th postovulatory day, a fluid filled space appears between the ectodermal layer and the cytotrophoblast which is called amniotic cavity. Its floor is formed by the ectoderm and the rest of its wall by primitive mesenchyme. The yolk sac appear on the ventral aspect of the bilaminar disk and is lined externally by the primitive mesenchyme and internally by the migrating endodermal cells from the endodermal layer of the germ disc

60. Formation of trilaminar embryonic disk: Fourteen days after fertilization, proliferation of ectodermal cells in the midline, leads to formation of primitive streak. Cells within the streak spread laterally between the ectoderm and endoderm as intraembryonic mesoderm. This intraembryonic mesoderm becomes continuous with the extraembryonic mesoderm at the lateral border of the embryonic disk.

61. Extraembryonic coelom: Extraembryonic mesenchyme, derived from the trophoblast appears to separate the yolk sac from the blastocyst wall and also the amniotic cavity from the trophoblast of the chorion. Small cystic spaces (lacuna) now appear within the extra-embryonic mesenchyme. These spaces gradually enlarge and fuse to form extraembryonic coelom. Progressive enlargement of the extraembryonic coelom, separates the amnion from the inner aspect of the chorion except at the caudal end of the embryo.

62. There, the mesenchymal attachment persists to form body stalk. Umbilical cord develops from this body stalk.

63. Subsequently the amniotic cavity enlarges at the expense of the extraembryonic coelom. The developing embryo bulges into the enlarged amniotic cavity. The yolk sac becomes partly incorporated into the embryo to form the gut. The part that remains outside is incorporated into the body stalk. Gradually the extraembryonic coelom is totally obliterated. The extraembryonic mesenchyme covering the amnion now fuses with the lining of the chorion. The single layer of fused amniochorion is now formed.

64. During the embryonic stage which extends from the fourth to eighth week, individual differentiation of the germ layers and formation of the folds of the embryo occur. Most of the tissues and organs are developed during this period. The embryo can be differentiated as human at 8th week.

65. However, the major structures which are developed from the three germinal layers are mentioned below. ECTODERMAL LAYER: Central and peripheral nervous system, epidermis of skin with its appendages, pituitary gland, chromaffin organs, salivary glands; mucous lining of the nasal cavity, paranasal sinus, roof of the mouth etc.

66. MESODERMAL LAYER: Bones, cartilage, muscles, cardiovascular system, kidney, gonads, suprarenals, spleen, most of the genital tract; mesothelial lining of pericardial, pleural and peritoneal cavity etc. ENDODERMAL LAYER: Epithelial lining of the gastrointestinal tract, liver, gallbladder, pancreas; epithelial lining of respiratory tract and most of the mucous membrane of urinary bladder and urethra; bulbourethral and greater vestibular glands etc.

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