ANP502: Reproductive Physiology OF FARM ANIMALS

1. O.A. Osinowo Department of Animal Physiology Federal University of Agriculture, Abeokuta, Nigeria ANP502:Reproductive Physiology OF FARM ANIMALS Introductory lecture compiled from various sources June 2014

2. Introductory lecture, covering: • Defining animal reproduction • Interdisciplinary nature of animal reproduction • Animal reproduction and economic livestock production • Role of reproduction in genetic improvement • Biological basis of sex • The chromosomal theory of sex determination • Genic balance theory of sex determination • Genetic dosage compensation / X-inactivation model of sex determination • Temperature dependent sex determination • Sex differentiation • Intersexuality • Molecular genetics of sex determination and differentiation

3. Topics to be covered by other lecturers: • Comparative anatomy of the male reproductive system. • Comparative anatomy of the female reproductive system. • Sexual development. • Gametogenesis • Spermatogenesis, sperm transport, sperm output, hormonal control of spermatogenesis; • Oogenesis, ova physiology, follicular development, ovulation, hormonal control of oogenesis. • Reproductive cycles. • Pregnancy. • Infertility in farm animals.

4. Defining Animal Reproduction • Animal reproduction: Process by which offspring are produced by male and female parents. • Involves heterosexual mating, conception, pregnancy, parturition and lactation. • Conception results from fusion of male and female gametes (i.e. spermatozoon and ovum), known as fertilization. • Animals must first attain reproductive age (puberty) before being capable of producing gametes. • Reproduction involves close coordination of various physiological events to be successful. This is usually achieved through the action of reproductive hormones.

5. Interdisciplinary nature of Animal reproduction • Proper understanding of animal reproduction requires knowledge in diverse fields such as: Reproductive physiology, molecular biology, endocrinology, environmental physiology, cell biology, immunology, genetics, biochemistry, sociology, reproductive diseases, psychology, embryology, obstetrics and so on.

6. Animal reproduction and economic livestock production • Livestock products such as milk and eggs are directly from reproductive processes. • Meat production depends on production of young animals (reproduction) for growing and fattening. • Profitability of livestock enterprise largely depends on reproductive efficiency.

7. Role of reproduction in genetic improvement Reproduction plays a major role in the genetic improvement of farm animals through the application of artificial insemination (AI) and multiple ovulation & embryo transfer (MOET). These help to increase selection differentials on the male and female sides respectively, leading to significant increase in the rate of genetic progress per year, as apparent from the equation below: DGy = SD * h2 GI where DGy= rate of genetic progress per year SD = selection differential h2 = heritability estimate, and GI = generation interval in years.

8. Sex = Sum total of those differences in structureand function on the basis of which an organism is classified as male or female. Sex determination & sex differentiation Sex determination is the initial event that determines whether the gonads will develop as testes or ovaries, while Sex differentiation refers to the subsequent events that ultimately produce either the male or female genotype. Theories of sex determination: Chromosomal theory Genic balance theory Genetic dosage compensation / X-inactivation model of sex determination Temperature dependent sex determination in alligators Biological Basis of Sex

9. The chromosomal theory of sex determination • According to this theory, sex is determined at fertilization by the sex chromosomes. • Each gamete contains the haploid number of chromosomes. • In mammals and in most insects, all gametes produced by the female are similar, having one X chromosome. • Males on the other hand produce two types of gametes in approximately equal numbers, one type bearing the X chromosome and the other type bearing the Y chromosome. • Hence in mammals and most insects, the female is known as the homogametic sex while the male is heterogametic.

12. The chromosomal theory of sex determination, contd. • At fertilization, the union of an egg with an X-carrying spermatozoon results in a zygote with 2 X chromosomes which develops into a female while the union of a Y-carrying spermatozoon with an egg results in an individual with one X and one Y chromosome, which develops into a male. • In birds, the female is the heterogametic sex while the male is homogametic (Table 2.1). • Chromosomal, genotypic or genetic sex is thus determined by the sex chromosomes received from the parents.

13. The chromosomal theory of sex determination, contd. • Aberrations of genetic sex do occur, either as a result of non-disjunction of sex chromosomes, translocation, deletion or mutation. • In maternal non-disjunction, fertilization of the ovum will lead to formation of either an XXX or an XXY zygote. In paternal non-disjunction, zygotes of XXY or XO genotypes result. Such cases of aneuploidy often result in gonadal or endocrine defects. • In man, the Klinefelter's syndrome in which the genotype is usually XXY instead of XY is characterized by gonadal hypofunction. • In women showing Turner's syndrome which is characterized by gonadal agenesis or aplasia, the genotype is frequently XO rather than XX.

14. Table 2.1 Kinds of chromosomal sex determination

15. Genic balance theory of sex determination • To some extent, genotypic sex may be considered an "all or none" or qualitative trait since usually male or female zygotes are formed at fertilization. • However, sex is a phenotypic trait, determined by interaction between genotype and environment. Individuals vary in their degrees of maleness or femaleness. • C.B. Bridges in 1922 proposed the genic balance theory to explain apparent quantitative variability in sexual character.

16. Genic balance theory of sex determination, contd. • According to this theory, sex is determined by the autosomes as well as by the X chromosomes, the ratio of X's to autosomes being the significant relation. • In Drosophila the X chromosome carries more genes for femaleness while the autosomes carry more genes for maleness (Table 2.2). • Which sex actually develops is decided by the balance between the two sets of genes.

17. Table 2.2 Sexual types in the fruit fly, Drosophila melanogaster

18. Genetic dosage compensation / X-inactivation model of sex determination • The theory of mammalian X-chromosome inactivation proposed by Lyon in 1961 holds that almost all the genes on one of the two X-chromosomes in the somatic cells of females are suppressed as a dosage compensation mechanism. • The choice of either the paternal or maternal X chromosome for inactivation is random. • The inactivated X chromosome is identifiable as it is heterochromatic and usually has its DNA replicated later in mitosis than other chromosomes. • The ZFY gene, thought to constitute the primary sex-determining signal, was identified within a very small segment of the Y chromosome by Page and co-workers in 1987. • However, the presence of a similar gene, ZFX, on the X chromosome prompted the propounding of the dosage compensation / X-inactivation theory of sex determination.

19. Genetic dosage compensation / X-inactivation model of sex determination, contd.  • According to this theory, both ZFX and ZFY produced functionally interchangeable proteins. • It therefore follows that XY cells would have two active copies of the gene while XX cells would only have one active copy, due to X-inactivation. • Embryos with two copies would thus develop into males while those with a single copy of the gene would develop into females. • In this way, the theory held that gene dosage determined sex. • Though elegant, the theory became untenable when it later became evident that the ZFX gene escapes X-inactivation, thus contradicting the dosage compensation model.

20. Temperature dependent sex determination • Many reptiles, including alligators and crocodiles, exhibit no sex chromosome dimorphism. • Rather, the sex of the offspring is determined by the temperature at which the eggs are incubated.   In alligators (Alligator mississippiensis), incubation of eggs from: 29 - 31.5oC result in 100% female offspring, 32.5 - 33oC result in 100% male offspring, 32oC 33.5 - 34.5oC different sex ratios, 35oC - 100% females Below 29oC and above 35oC, high mortality result. In turtles, males are generally produced at lower temperatures than females: 22.5-27oC result in mostly male turtles Around 30oC only female turtles arise

21. Sex Differentiation • Sex differentiation is the process by which the reproductive system of an animal develops into either that of a male or a female during embryogenesis and foetal development.

22. Sex Differentiation ...contd. • Gonadogenesis is the process by which the gonads are formed during foetal development. • Genital or gonadal ridge from which the gonads and reproductive ducts develop are formed early in organogenesis. • The primordial germ cells originate extragonadally near the yolk sac endoderm. • They migrate by amoeboid movement to the genital ridge. Their failure to arrive leads to gonadal agenesis while their arrival leads to formation of the gonads.

23. Sex Differentiation ...contd. • If the individual is to be a male, the germ cells give rise to the seminiferous tubules. • If the individual is to be a female, additional migration and proliferation of epithelial and germ cells occur which give rise to the ovarian cortex. • The reproductive systems of both males and females undergo a stage of embryonic development known as the indifferent stage: • At this stage all the rudiments of male and female reproductive systems are present and it is difficult to tell the sexes apart.

24. Sex Differentiation ...contd. • Development of the mesonephric (or Wolffian) duct and the paramesonephric (or Mullerian) duct follow that of the gonad closely. • Usually one duct persists while the other regresses. • Arising from their common origin in the indifferent stage, homologues of the male and female reproductive systems are recognizable. • In typical development the genetic sex, gonadal sex and the sex of the accessory organs are the same.

25. Indifferent Stage of Mammalian Sexual Development Indifferent Stage Female Male Undifferentiated Gonads Testis Medulla Ovary Cortex Wolffian duct Mullerian duct Male Duct System Embryonic Ducts Female Duct System

26. Table 3.1Homologues of the Male and Female Reproductive System

27. Intersexuality • An intersex is an animal with congenital anatomical defects which confuse the diagnosis of sex. • Best known example is the bovine freemartin • Observation that female co-twin in cows is often sterile first made by Varro (100 BC) and Columella (AD 100)

28. Intersexuality ... Contd. • Freemartinism is the sexual modification of a female twin by exchange of blood with a male foetus during pregnancy in cattle. • Results from the fusion of the chorioallantois of adjacent embryos during multiple pregnancy. • Blood forming cells and other cells are exchanged between the foetuses. • In 5% of bovine twin pregnancies no fusion of the chorioallantois occurs and the female is not a chimera and is fertile.

29. Characteristics of Freemartins • Presence of internal reproductive organs of both sexes • Modified ovaries with varying degrees of similarity with the testis • External genitalia like those of a normal female • Blood chimerism (Blood group & Sex chromosomes)

30. MOLECULAR GENETICS OF SEX DETERMINATION AND DIFFERENTIATION Chromosomes associated with sex determination • The differentiation of the bipotential gonads into either testes or ovaries depends on the interplay of genes located on sex chromosomes (X and Y) and autosomal chromosomes. • The Sry gene, located on the Y chromosome, encodes the transcription factor, SRY, which is responsible for triggering the indifferent gonads to develop as testes rather than ovaries.

31. MOLECULAR GENETICS OF SEX DETERMINATION AND DIFFERENTIATION … contd. • Current research suggests that both male and female pathways rely on dominantly acting genes, with SRY actively promoting the male pathway by upregulating SOX9 expression, while b-catenin, Rspo1, and Foxl2 actively promote the female pathway by repressing SOX9: • Which pathway prevails is a matter of timing (and expression level).

32. MOLECULAR GENETICS OF SEX DETERMINATION AND DIFFERENTIATION … contd. Gene symbols • In humans:Gene symbols generally are italicized, with all letters in upper case, e.g. SRY. • Protein designations are same as the gene symbol but are not italicized, with the first letter in upper case and the remaining letters in lower case, e.g. Sry. • In mouse and rat: gene symbols generally are italicized, with only the first letter in upper case and the remaining letters in lower case, e.g. Sry. • Protein designations are the same as the gene symbol but are not italicized and all are upper case, e.g. SRY.

33. MOLECULAR GENETICS OF SEX DETERMINATION AND DIFFERENTIATION … contd. Transcription factor: • A protein that binds to specific DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to messenger RNA. • Transcription factors perform this function alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase to specific genes.

34. Genes associated with sex determination

35. Genes associated with sex determination … contd. [Sources:www.genecards.org, www.en.wikipedia.org, www.ncbi.nlm.nih.gov ]

36. MOLECULAR GENETICS OF SEX DETERMINATION AND DIFFERENTIATION … contd. Differentiation of indifferent gonads into testes • SRY is at the top of a genetic cascade that directs the differentiation of the bipotential gonads into testes through the activation of the Sox9 gene which is its direct target. • Sox9 gene encodes the transcription factor, SOX9, a protein which acts during chondrocyte differentiation, and, with steroidogenic factor 1 (Sf1), regulates transcription of the anti-Mullerian hormone (AMH) gene.

37. MOLECULAR GENETICS OF SEX DETERMINATION AND DIFFERENTIATION … contd. Differentiation of indifferent gonads into ovaries • SOX9 also plays a pivotal role in male sexual development; working with Sf1, it produces AMH in Sertoli cells to inhibit the creation of a female reproductive system. • It also interacts with some other genes to promote the development of male sex organs.

38. MOLECULAR GENETICS OF SEX DETERMINATION AND DIFFERENTIATION … contd. • Genes involving Wt1 and Sf1 are involved in the establishment of the genital ridge and their early expression probably initiates Sox9 expression in both males and females. • b-catenin probably begins to accumulate in response to Rspo1 – Wnt4 signaling. • Sufficient accumulation of b-catenin represses SOX9 activity. • Later, FOXL2 levels increase, helping to maintain granulosa (follicle) cell differentiation by repressing Sox9 expression.

40. MOLECULAR GENETICS OF SEX DETERMINATION AND DIFFERENTIATION … contd. Maintenance of testis and ovary • Maintenance of the structure and function of the testis and ovary is a life-long event, to suppress the potential for transdifferentiation. • A single factor, the transcriptional regulator, FOXL2, is required to prevent transdifferentiation of an adult ovary to a testis. • Inducible deletion of Foxl2 in adult ovarian follicles in the mouse leads to immediate upregulation of testis-specific genes including the critical SRY target gene Sox9.

41. MOLECULAR GENETICS OF SEX DETERMINATION AND DIFFERENTIATION … contd. • Concordantly, reprogramming of granulosa and theca cell lineages into Sertoli-like and Leydig-like cell lineages occurs with testosterone levels comparable to those of normal XY littermates. • In like manner, loss of the DMRT1 transcription factor in mouse Sertoli cells, even in adults, activates Foxl2 and reprograms Sertoli cells into granulosa cells. • In this environment, theca cells form, estrogen is produced and germ cells appear feminized. • Thus, Dmrt1 is essential to maintain mammalian testis determination.

42. Figure 2. Model of Sox9 regulation required for maintenance of gonadal phenotype in mammals. Source: Henriette Uhlenhaut et al. (2009)

43. Reading list Alaku, S.O. 2013. Physiology of Reproduction in Animals of Economic Importance. Jully Prints, Enugu, 398 pp. Anon, 2013. The genetics of sex determination: Rethinking concepts and theories. In: Schiebinger, L., Klinge, I., Sanchez de Madariaga, I. and Schraudner, M. eds. (2011-2013). Gendered innovations in science, health and medicine, engineering and environment. http://genderedinnovations.stanford.edu.case-studies/genetics.html Uhlenhaut, N.H., Jakob S., Anlag, K., Einsenberger T., Sekido, R., Kress, J., Treier, A., Klugmann, C., Klassen, C., Holter, N.I., Reithmacher, D., Schutz, G., Cooney, A.J., Lovell-Badge, R. and Treier, M. 2009. Somatic sex programming of adult ovaries to testes by FOXL2 ablation. Cell 139: 1130-1142. Matson, C.K., Murphy, M.W., Sarver, A.L., Griswold, M.D., Bardwell, V.J. and Zarkower, D. 2011. DMRT1 prevents female reprogramming in the postnatal mammalian testis. Nature, 476:101–104. Osinowo, O.A. 2006. Introduction to Animal Reproduction. Sophie Academic Publishers, Abeokuta, 91 pp. Veitia. R.H. 2010. FOXL2 versus SOX9: A lifelong “battle of the sexes”. Bioessays, 32: 375-380. Wikipedia, 2013. Temperature dependent sex determination. http://en-wikipedia.org/wiki/Temperature-dependent_sex_determination.