Advances in the treatment of infertility

Till few years back options for the treatment of fertility were limited but, with the advances in assisted reproductive techniques (ART) the couples who were termed as sterile were able to procreate. ART consists of various methods that are used to manipulate oocytes and sperm to overcome infertility. The first successful artificial insemination was done in early 1770’s and the first In vitro fertilization (IVF) was done by Steptoe and Edwards in 1983. In 1992 intra cell sperm injection (ICSI) of a single sperm into an oocyte was performed by Palermo et al. IVF is said to be the backbone of ART and in the last 20 years lot of advances have been made in the field of assisted reproductive technology (ART) viz.

Superovulation:

The hormonal intervention by which the ovulation is increased during ovulatory cycle due to which multiple oocytes are released is termed as superovulation. For the stimulation of ovaries; exogenous human gonadotropins are used which have FSH and LH activity e.g. human menopausal gonadotropin (HMP). Recombinant FSH and LH are also used for ovarian stimulation these days. Ovarian stimulation with gonadotropins is combined with Gonadotropin releasing hormone (GnRH) agonist e.g. leuprolide acetate so that the endogenous LH is suppressed and premature leutinization of follicle is avoided.

Oocyte retrieval:

Oocytes are retrieved transvaginally under the guidance of ultrasound after sedating the patient 34-36 hours of hCG administration. This timing is important for the maturation ofoocyte and also to minimize the spontaneous ovulation. After retrieval, oocytes are separated from the follicular fluid and are separated according to morphology and mature oocyte which is characterized by the presence of first polar body is separated as these are best for fertilization in vitro.

In vitro maturation (IVM):

In this process, the immature oocytes are retrieved from the unstimulated ovaries by aspirating them from antral follicles during the early phase of menstrual cycle under the guidance of an ultrasound. After retrieving, eggs are matured in the laboratory for 24-48 hours. After maturation these eggs can be used for fertilization using ICSI etc. IVM is very helpful in patients having polycystic ovarian syndrome (PCOS) and for preserving fertility in cancer.

Embryo transfer (ET):

For embryo-transfer a sample of sperm is prepared on the same day of oocyte retrieval. This sperm sample is processed by percoll gradient centrifugation technique and is incubated in protein rich medium for 4 hours to initiate capacitation. Now, the mature oocytes are inseminated and are examined after 18 hours to check fertilization which is confirmed by the presence of 2 pronuclei and 2 polar bodies in the perivitelline space. After 72 hours when embryo reaches 4-8 cell stage; they are transferred into the uterus.

Microsurgical epididymal sperm aspiration (MESA):

It is an epididymal sperm retrieval technique in which each epididymal tubules are identified under operating microscope and sperms are aspirated or micro punctures are made for obtaining a fine amount of sperm. Alternatively, tubules can be incised and fluid can be gathered. As sperms in the epididymal fluid are highly concentrated therefore, sperms are retrieved in microliters only. The retrieved sperms can be used immediately or cryopreserved.

Percutaneous epididymal sperm aspiration (PESA):

In PESA, epididymal sperms are aspirated without surgical opening of scrotum. The patient is given general anaesthesia (GA) and a butterfly needle attached to a 20ml syringe is inserted into the caput epididymis assuming that sperm must be there and sperms are aspirated and the needle is withdrawn gently. The aspiration is done repeatedly till surgeon gets sure of the amount of epididymal fluid.

Testicular sperm aspiration (TESA):

In this technique, patient is given GA and a small incision is made in the scrotal skin and with the help of spring loaded needle sperms are collected from the testes.

Testicular sperm extraction (TESE):

In this process, under GA a small portion of tissue from the testes is removed and viable sperm cells are extracted from that tissue. The amount of sperm obtained is low and any injury to testes can lead to testicular infarction.

Testicular sperms can also be retrieved during Vasovasostomy and Vasoepididymostomy if motile sperms are found during the operative procedure.

Intra cytoplasmic sperm injection (ICSI):

It is an invasive microinjection technique in which single sperm is directly    injected into the cytoplasm of a mature oocyte. During ICSI, the microinjection pipette containing the single sperm pierces the zonapellucida at 3 o’clock position and is pushed deep into the cytoplasm; sperm is injected and needle is removed. After 16 hours of injection, oocyte is examined for fertilization and if successful, embryo is transferred after 1-3 days of fertilization. ICSI has replaced largely subzonal sperm injection (SUZI), partial zona dissection (PZD) and zona drilling (ZD).

Pre-implantation genetic diagnosis (PGD):

PGD allows identification of specific genetic disorders before embryo transfer. For PGD, the embryo at 8-cell stage is used for biopsy and during biopsy the zonapellucida is pierced and a single blastomere is removed for analysis. Along with blastomere biopsy, polar body and trophectoderm biopsy can also be done. For chromosome analysis fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are used and for identification of whole chromosome abnormalities; comparative genomic hybridization (CGH) is used.

Assisted hatching:

Inability of blastocysts to hatch from zonapellucida is one of the main factors responsible for implantation failure of IVF. In this technique, the embryo to be transferred is held using a micropipette and with the help of acidic solution or laser or other mechanical methods; a small hole is created in the zonapellucida. The embryo is then washed to get rid of the medium and is placed in incubator until ET.

Cryopreservation:

In this technique, cells or tissues are preserved at sub-zero temperature. Now days, apart from embryo and sperm preservation; oocytes and ovarian tissues can also be cryopreserved in women with risk of premature ovarian failure e.g. in women with Turner’s syndrome. For cryopreservation, ovarian cortex is isolated and cut into strips of 10mmx5mmx1mm dimensions and are then incubated with cryoprotectants e.g. dimethyl sulfoxide (DMSO) and are frozen using programmable freezer.

Vertification:

It is an alternative method of preservation using the benefits of cryopreservation. Cryopreservation leads to ice formation but, in vertificationcryoprotectants are added prior to cooling and thus by rapid cooling ice formation is avoided and cells are stored in a glass state. Process of vertification is still experimental.

                                                     Dr.Bharati Sood

Cytokines and Pregnancy

Evidence shows that cytokines are produced in reproductive and embryonic tissues also and are involved in the invasion of trophoblasts and that invasion is important for regulation and maintenance of pregnancy. Also, cytokines play crucial roles in implantation of embryo during early stages of pregnancy and spiral artery remodelling, regulation of trophoblast invasion, immunoregulation and initiation of labour during later stages of pregnancy. More recently it is being shown that cytokines are produced by cells in the decidua and placenta also apart from immune cells. Cytokines and chemokines are secreted from cytotrophoblasts as well as from the cells of decidua predominantly from macrophages and fibroblasts. Also their receptors have been identified on cytotrophoblasts. It has been seen that chemokines such as CX3CL1, CCL4, CCL14, and CXCL16 increase trophoblast migration or invasion. Similarly, interleukins like IL-1, IL-6, IL-8 and IL-11 which are secreted by cytotrophoblast and decidual natural killer cells promote gelatinase activity and/or promote trophoblast invasion. More and more growth factors have been identified playing key roles in trophoblast invasion but, it is still not understood fully whether some play prominent role than others or not.

Interleukin-1 (IL-1) is a polypeptide which is generally produced after inflammation, injury or an antigenic challenge by mononuclear phagocytes. IL-1is also synthesized in epidermal, epithelial, lymphoid and vascular tissues by keratinocytes, fibroblasts, B lymphocytes, natural killer cells, astrocytes, microglial cells of brain etc. Initially IL-1 was called as endogenous pyrogen as it was thought to cause fever but, later it was found that it does more than just causing fever[Dinarello 1988]. IL-1 family is a group of pro-inflammatory cytokines which consists of two ligands or agonist proteins IL-1α and IL-1β, their cell surface receptors interleukin-1 receptor type 1 (IL-1R1) and interleukin-1 receptor type 2 (IL-1R2), a non-binding receptor accessory protein (IL1RAcp) and the receptor antagonist (IL-1ra). This naturally occurring IL-1ra competes with IL-1 for receptor binding. Evidence shows that all components of the IL-1 family have been identified in the human endometrium and also, that IL-1 is present at the feto-maternal interphase. IL-1 is produced by cells of trophoblast as well as decidua and IL-1 is known to have an important role in implantation. Research on mice has shown that IL-1 has a potential role in embryo implantation as IL-1 receptor antagonist given before implantation leads to the reduction in the number of implanted embryos. IL-1 plays an essential role in onset and development of invasion which is required for blastocyst implantation and placentation and this is highly regulated by a complex system of inhibitors consisting of receptor antagonist IL-1ra and IL-1 receptor (IL-1R_I). Evidence shows that IL-1 is essential for initiating as well as maintaining adequate invasion. IL-1 when produced at the site of invasion causes stimulation of surrounding cells which leads to the synthesis and release of chemokines as well as other cytokines e.g. IL-2, IL-4 etc. causing activation of cells. During early pregnancy, IL-1 is mainly produced and secreted by maternal decidua. In the endometrium, IL-1 is produced by macrophages, glandular epithelium and stromal cells in endometrium. Invasive properties of trophoblastic cells are related to their capacity of secreting proteolytic enzymes such as matrix metalloproteinases (MMPs). IL-1 is thought to promote trophoblast invasion by stimulating MMP-9 release by human cytotrophoblast.

Interleukin-6 (IL-6) is a glycosylated polypeptide consisting of 184 amino-acids with a molecular weight of 21-28 KDa approximately.IL-6 has a four helix bundle type tertiary structure and is manufactured by a diverse number of cells in different tissues like adipose tissue, skeletal muscle, liver etc. IL-6 is a cytokine that performs multiple functions and plays an important role in immune response, host defence mechanism, regulation of haematopoiesis, acute phase reaction as well as both anti and pro inflammatory events. IL-6 exhibits its activity by binding to its membrane bound receptor known as IL-6 receptor (IL-6R). This binding of IL-6 to IL-6R causes dimerization of the signal transducing receptor subunits glycoprotein 130 (gp130). This is followed by recruitment of IL-6R complex which initiates a series of cascades. Evidence shows that mice deficient in IL-6 have reduced fertility and decrease in viable implantation sites which shows that IL-6 plays an important role in implantation. IL-6 mRNA and protein are localized in human endometrium as well and IL-6 is expressed maximum during mid-secretory phase, thus, showing importance at the time of implantation. Moreover, the secretion of IL-6 by endometrial stromal cells is accelerated by IFN-γ. It has been seen that during first trimester IL-6 mRNA and protein are present in cells of cytotrophoblast as well as syncytiotrophoblast and also, the concentration of IL-6 is higher in decidua when compared to placental tissue. As IL-6 is present at the feto-maternal interphase, it point towards its involvement in the process of trophoblast invasion which is proved also. Tumour invasion and invasion of trophoblasts share the common biochemical mediators which are MMPs and their tissue inhibitors (TIMPs). For implantation and placentation to take place massive invasion by the cells of trophoblast is essential as well as required and the invasive activities of trophoblasts are related to their ability to secrete proteolytic enzymes such as MMPs. The process of invasion is controlled very well in terms of location as well as developmental stage. Furthermore, IL-6 is known to have positive effect on the production of these MMPs by regulating their production. 

Tumour necrosis factor alpha (TNF-α) also known as cachectin is a member of TNF superfamily which consists of 8 ligands and 29 different receptors that are involved in various cellular processes produced mainly by activated macrophages and to a lesser extent by activated mast cells and natural killer (NK) cells. It is also considered as an inflammatory cytokine due to its similar activities to IL-1.TNF-α exhibits a wide range of its biological properties via two distinct receptors termed as TNFR1 and TNFR2 which are members of the TNF receptor superfamily. TNF is known to control the expression of other cytokines, immune receptors, proteases, growth factors and cell cycle genes which in turn regulate inflammation, cellular homeostasis, apoptosis, cell survival, proliferation, differentiation and migration. In situ hybridization studies and immunohistochemistry analysis has shown that TNF mRNA proteins are expressed in the endometrium as well as the smooth muscle cells of the myometrium. Furthermore, different cell types of the endometrium viz. glandular epithelial cells, macrophages, vascular cells and fibroblasts express TNF. TNF mRNA is seen to be expressed in all cell types of trophoblastic lineage which includes villous cytotrophoblasts, syncytiotrophoblasts, proliferating cytotrophoblasts of cell islands and invasive trophoblasts during the first third of gestation but, as pregnancy proceeds the expression of mRNA is switched from the population of trophoblastic cells to villous stromal cells. This is maintained even when extravillous trophoblasts cause the endothelial cells displacement of spiral arteries. However, in the later stages of pregnancy, the expression of TNF is decreased in the invasive cells and in the trophoblast giant cells; expression of TNF is totally absent. However, some researchers have shown that TNF specifically inhibits trophoblast migration and invasion but, despite its negative effects on invasion; TNF was shown to stimulate the production of MMPs specifically MMP-9 in the first trimester trophoblast explant cultures and decidual cells.

Lipopolysaccharide (LPS) is commonly found as major constituent of cell walls of gram negative bacteria. The outer layer of gram negative bacteria is phospholipoprotein bilayer of which the outer leaflet is lipopolysaccharide. LPS is extremely toxic and is termed as endotoxin. LPS consists of a toxic lipid A, a core polysaccharide and O antigen polysaccharide side chains. Lipopolysaccharide helps in maintaining the membrane structure and protection from various types of chemicals. Evidence shows that LPS can increase the production of MMPs in human foetal membranes. Also, it causes preterm birth by effecting the stimulation of cytokines and prostaglandins by host cells. Almost all MMPs are released as pro MMPs; the inactive precursor form. Hence, activation of these pro MMPs is an important factor for MMP activity to occur. The most common mechanism that has been recognised for activation of MMPs is proteolytic removal of a propeptide domain of pro MMPs which is done by a number of endogenous proteinases such as, plasmin, trypsin and other active MMP family membranes. These endogenous proteinases then form MMP-activating cascades in vivo. At the site of infection, various types of proteinases are released by pathogenic bacteria such as gram negative bacteria and LPS is a component of gram negative bacteria. Therefore, at the site of infection, bacteria induced inflammation may cause the activation of pro MMPs and bacteria by means of LPS causing degradation of matrix by upregulating MMP production and converting pro MMPs into active MMPs. The presence of infection during pregnancy is attributed to gram negative and gram positive bacteria which leads to some serious consequences such as preterm premature rupture of membranes (pPROM), preterm labour (PTM) etc. and the molecular mechanisms of PROM involves the activation of MMPs in the amniochrion. Hence, knowing how and in what way LPS stimulates MMP production will be useful.

Matrix metalloproteinases (MMPs) are proteolytic zinc requiring enzymes which consists of many different types such as Collagenases viz. MMP-1, 8, 13, 14 and 18; the Stromelysins viz. MMP-3, 10 and 11; Gelatinasesviz. MMP-2 and 9 and Matrilysins viz. MMP-7 and 26 and are responsible for degradation of extracellular matrix. During placentation gelatinases play an important role in the process of invasion by the trophoblast cells into the maternal endometrium. Development of placenta is a complex process which requires a perfect coordination between the maternal endometrium and foetal-derived trophoblast cells. The process of invasion begins when blastocyst adheres to the endometrial epithelium and during invasion the properties of invasive placental cells are similar to malignant cells but, placental cells are highly regulated. The mechanism of invasion has been studied a lot by researchers and it has been found that the ability of cytotrophoblast cells to cross the basement membrane and interstitial matrices point towards the presence of some matrix degrading enzymes which play an important role in invasion which are none other than MMPs. There is evidence that human placental trophoblast cells produce matrix proteins such as collagen IV, laminin and fibronectin in first trimester. MMP-2 which is a 72KDa and MMP-9 which is a 92KDa type IV collagenase; are the main enzymes that cause the degradation of the basement membrane which consists of type IV collagen chiefly. The activity of gelatinases also relies on the activity of other proteases which cause the transformation of pro MMPs to active MMPs. After blastocyst implantation and before the completion of placentation in first trimester trophoblast cells produce both MMP-2 and 9 but, MMP-2 dominates while MMP-9 is produced in minor amount whereas, in the third trimester cells of trophoblast secrete MMP-9 predominantly when compared to MMP-2. Physiologically, the activity of these MMPs is controlled by tissue inhibitors of metalloproteinases (TIMPs). Three tissue inhibitors viz. TIMP-1, TIMP-2 and TIMP-3 regulate proteinase activity. TIMPs inhibit the activity of different types of MMPs by binding to the zinc binding site of active MMP. TIMP-1 forms complexes and mainly regulates MMP-9 whereas, TIMP-2 with MMP-2. TIMP-3 is known as main regulator of MMPs in vivo as it supports the activation and inhibition of MMP-2. Evidence shows that TIMPs are produced throughout the pregnancy by decidual and trophoblast cells. Hence, trophoblast invasion requires MMP-2 and MMP-9 synthesis as well as activation.

Cytokines in pregnancy disorders

The process of invasion begins when the EVT starts to invade the decidualized endometrium and inner myometrium. This is followed by transformation of maternal spiral arteries into low resistance vessels as described before. Any failure in the remodelling of spiral arteries causes a decrease in uteroplacental blood flow. This poor perfusion is thought to be responsible for the release of cytokines and products of oxidative stress into maternal circulation, which may cause endothelial dysfunction. Growth factors and cytokines are responsible for blastocyst implantation, trophoblast invasion and spiral artery remodelling. Research shows that cytokines play an important role in the process of invasion of trophoblast cells by regulating the secretion of gelatinases. Furthermore, this poor or incomplete transformation of vessels has been detected in placental bed of patients having pre-eclampsia and IUGR which indicates that EVT differentiation may be disturbed in these patients. Hence, decreased invasion leads to malformation, IUGR, pre-eclampsia, spontaneous abortion, still birth etc. whereas, increased invasion is known to cause invasive mole, placenta accreta and choriocarcinoma. Therefore, the processes of invasion are of great importance for achieving normal pregnancy and as cytokines are known to regulate trophoblast invasion by regulating MMP-2 and MMP-9, any finding on how cytokines regulate invasion and what effect cytokines have on the process of invasion will be very helpful.

                                                     Dr.Bharati Sood

Understanding Growth factors and Cytokines

Growth Factors-

Growth factors are proteins or steroid hormones that bind to receptors on cell surface and promote cellular growth, proliferation and differentiation. These can also be defined as the extracellular signalling molecules that are involved in cell-cell communication. Growth factors are known to regulate cellular processes by signalling information between the various cells. Growth factors are thought to have evolved in order to perform cell-cell communication in multicellular organisms. This cell-cell communication occurs by cell signalling which is a system of communication by which cells detect and respond to any external stimuli present. Cells respond to chemical signals which are primarily synthesized and released by adjacent cells but, signals can be anything from light, temperature, touch, pH etc. The cells response according to the stimuli and the response were initially thought to be growth and proliferation hence, the term growth factor was used but, studies have shown that growth factors are not only important for growth and proliferation but, also for cell differentiation, survival, division, synthesis, transformation, cell death and motility depending upon the stimuli. Most of the growth factors act on neighbouring target cells by binding to specific high affinity plasma membrane receptors [Thomas et al 2008]. This binding to specific receptors causes growth factors to induce signal transduction pathways which leads to the activation of effector mechanisms present within the cell that responds. Nerve growth factor (NGF) was the first growth factor being isolated and studied [Cohen & Levi-Montalcini 1957] soon followed by epidermal growth factor [Cohen 1965].Growth factors are named majorly on the basis of the cell type or the tissue from which the factor was first isolated e.g. Platelet derived growth factor (PDGF) and Brain derived growth factor (BDGF) or on the basis of the response that is elicited in the targeted cell upon receptor binding e.g. Hepatocyte growth factor (HGF) and Fibroblast growth factor (FGF) or on the basis of the main action that is stimulated e.g. Transforming growth factor (TGF) and Bone morphogenetic factor (BMP).[Thomas et al 2008]

Cytokines-

Initially, cytokines were defined as the extracellular signalling proteins that were responsible for interaction with the immune cells while growth factors were thought to act on other cells. Hence they were collectively grouped as immunocytokines or immunokines. But, now evidence shows that these signalling proteins mediate and regulate inflammation, haematopoiesis and other cellular processes as well apart from immunity and hence, are termed as cytokines [Thomas et al 2008]. Cytokines are produced by cells other than immune cells also. They are also produced by glial cells of the nervous system. Different types of cells respond in different manner to variety of cytokines. Sometimes, the terms growth factors and cytokines are used interchangeably as clear demarcation is not possible due to diversity of their functions. Interleukins (IL), Growth hormone, Tumour necrosis factor (TNF) and interferons (INF) comprise main cytokines. Cytokines belong to a unique family of growth factors which are secreted mainly from leukocytes. Those secreted from lymphocytes are termed as lymphokines while the ones secreted by monocytes or macrophages are known as monokines. Many lymphokines are called as interleukins as they are not secreted by leukocytes but, affect cellular responses of leukocytes and chemokines are cytokines having chemotactic activities. Cytokines are known to activate phagocytic cells and stimulate humoral as well as cellular immunity. Apart from regulating immunity, haematopoiesis and inflammation, cytokines are now known to be involved in neurogenesis, oncogenesis and embryonic development of an organism. Moreover, cytokines influence the actions of other cytokines and act in cascades [Naruse K et al 2010].

The mechanism of action of cytokines is by paracrine signalling i.e. a target cell is in the vicinity of emitting cell, autocrine signalling i.e. when a signal targets the cell itself and juxtacrine signalling in which signals target adjacent cells through a synapse [Thomas et al 2008]. It is known that traditional cytokines like IL-1 and IL-6 are secreted by non-immune cells whereas, growth factors like FGF and TGFβ-2 are secreted by cells of immune system. IL-1 is an inflammatory cytokine which is produced by activated macrophages and it is known to promote activation and secretion of cytokines and other acute-phase proteins. IL-2 is primarily produced by T helper cells. It is the major growth factor for T cells and it promotes the growth of B cells and can activate NK cells and monocytes. IL-4 is produced by T cells and mast cells.IL-5 is a cytokine produced by Th2 cells and it promotes the growth and differentiation of B cells and eosinophiles. IL-7 is responsible for apoptosis of tumour cells and causes differentiation of cells from a subgroup of acute myeloblastic leukaemia. IL-8 acts as a chemotactic factor that attracts neutrophils, basophils and T-Cells to sites of inflammation. IL- 10 is mainly an inhibitory cytokine which is produced by activated macrophages, B cells, and T helper cells. TNF-α is an important mediator of acute inflammation and is produced by activated macrophages in response to microbes, for example reaction to the presence of the lipopolysaccharide (LPS) of Gram negative bacteria. Interferon-γ (IFN-γ) is an important cytokine produced by Th1 (T helper type 1) cells and NK cells. IFN-γ has numerous functions in both innate immune and adaptive immune systems. 

Chemokines are chemotactic cytokines produced by many kinds of leukocytes and other cell types. Chemokines recruit leukocytes to sites of infection and play a role in lymphocyte trafficking. Transforming growth factor-β (TGF-β) is a cytokine produced by T cell, macrophages, and many other cell types. It is an inhibitory cytokine that inhibits the proliferation of T cells and blocks the effects of pro-inflammatory cytokines.TNF-α, IL-1, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, and chemokines play an important role in the innate immune system. Though the terms growth factors and cytokines are used interchangeably but, the term growth factor is used with respect to proliferation of cells whereas, cytokine is a neutral term which means it can cause cells to proliferate, differentiate, and divide or any other effect. [Kayisli et al2009][Elgert 1996][Naruse K et al 2010]

                                                               Dr.Bharati Sood

Placental Development

Introduction:  

Placenta is a very important organ for the growth and development of the embryo and foetus. It forms a connection between the blood supply of the embryo and the blood supply of mother; though the connection is being separated by thin membranes. Placenta is an essential site for the gaseous, waste and nutrient exchange between the foetus and the mother. Apart from this it synthesizes hormones that maintain and regulate pregnancy. Development of placenta is a highly regulated process that is initiated after the implantation of blastocyst in the endometrium. The outer layer of blastocyst becomes the trophoblast that has an important role in the implantation and development of the embryo and foetus. It develops gradually into the placenta and provides nutrition to the foetus. Invasion of the placenta by the trophoblast and spiral artery re-modelling is essential for further development of placenta. Therefore, it is very important to know about the whole complex process of placental development as any deviation from the normal process of placentation may lead to serious consequences. [Dr.Hills 2010]

Development of placenta:

Differentiation of trophoblast:

The development of placenta begins when the blastocyst attaches itself to the maternal endometrium. The outer layer of the cells that form the blastocyst is termed as trophoblast and this is the first extra-embryonic cell lineage differentiation. By the 6-7 days after fertilization, this trophoblast invades the endometrium and gets differentiated into two layers viz. an inner cytotrophoblast and outer syncytiotrophoblast [Fig.1]. Other cytotrophoblast cells differentiate into extravascular trophoblast (EVT) and this EVT plays a very crucial role in re-modelling of spiral arteries. Cytotrophoblast is mononuclear and is active mitotically whereas syncytiotrophoblast is multinucleated and is derived from cytotrophoblast. The cytotrophoblast keep on making new trophoblast cells. Various factors are responsible for regulating this trophoblast differentiation in humans like oct-4, Hash-2, FGF-4, Id-2, Stra 13, H1F1a, Gcm-1, BMP, EGF, PPARγ, DIx-4, SDF-1, E-factor, TNFα etc. There must be synchronization between the implantation and placentation by the blastocyst. The endometrial extracellular matrix consists of proteins to which trophoblast attaches and with the help of proteases causes the degradation of these proteins. [Varney et al. 2004]

Fig.1 Differentiation of trophoblast into cytotrophoblast & syncytiotrophoblast [cited in Hills 2010]

Invasion of trophoblast and development of villous structure:

The trophoblast starts to invade the endometrium after 6-7 days of fertilization by protruding the endometrial stroma with finger-like projections. From syncytiotrophoblast finger-like projections arise which penetrate into the endometrial stroma through the endometrial epithelium [Fig.2]. Endometrial stroma consists of glands and capillaries. When syncytiotrophoblast invades the stroma around 8^th day, lacunae or hollow spaces start forming leading to the erosion of endometrial glands and rupture of capillaries. These capillaries fill the lacunae with embryotropin which is a mixture of secretions from the glands and maternal blood. This provides nutrition to the embryo. Around 12^th day post fertilization the lacunae in the syncytiotrophoblast join together to communicate with each other and the fused lacunae develops into intervillous space (IVS). The IVS is not filled with highly oxygenated maternal blood until the establishment of foetal vessels in villi. Therefore, the placenta remains in a hypoxic environment in initial months. The endometrium undergoes decidual reaction at the same time. The cells of the stroma become plump by accumulating glycogen and lipids and are known as decidual cells. The capillaries become dilated to form sinusoids which undergo erosion by the invading trophoblast to fill the lacunar network with blood. [Blackburn 2007]

Fig.2 showing invasion of trophoblast [cited in Reproductive biology 2010]

Decidualization of the endometrium plays an important role in protection of the foetus and regulation of placentation. Decidua consists of three areas viz. decidua basalis (part that lies below the site of implantation of embryo), Decidua capsularis (part overlying implanted embryo) and Decidua Patietalis (remaining part of the decidua) [Fig.3]

Fig.3 showing various parts of decidua [cited in Nursing crib.com 2010]

Cytortophoblast differentiates into vascular cytotrophoblast and the extravascular trophoblast. The vascular cytotrophoblast fuses to form chorionic villi and extravascular cytotrophoblast is responsible for the remodelling of spiral arteries. Chorionic villi start to form 14 days post fertilization when cytotrophoblast arranges itself into column of cells that extend into the syncytiotrophoblast and thus form the primary villi. The primary villi start branching and are then termed as secondary villi. The mesenchymal cells that are present in the secondary villi start to form blood vessels in the villi and these blood vessels are connected with the blood vessels of chorionic sac and to the embryo through the umbilical cord. These villi are termed as tertiary villi now [Fig.4]. The villi that are attached to the decidua basilis are termed as anchoring villi. These villi consist of arteries, veins, arterioles and venules. The villi that grow from the sides of the anchoring villi and project into intervillous space are termed as intermediate villi. Intermediate villi branch and form terminal villi which contain dilated capillaries.

Fig.4 showing primary, secondary & tertiary villi [cited in Anatomy, University of Michigan Medical School 2000]

The cytotrophoblast cells at the end of the villi penetrate deep into the endometrium and around 8^th week post fertilization these villi cover the whole of the area of the chorionic sac. The villi present in the decidua basilis keep on proliferating forming a tree-like structure called as chorionic frondosum. Development of villous structure and angiogenesis is stimulated by VEGF (vascular endothelial growth factor), placental-like growth factor and by hypoxic environment. The villi that are present near the decidua capsularis degenerate as blood flow to these villi decrease due to the compression of decidua capsularis. This result in an avascular area called as chorion leave. It fuses with the decidua vera and forms chorion (outer membrane) and amnioblasts of the cytotrophoblast forms amnion (inner membrane). The villi near the decidua basilis rapidly increase their number and have more blood and mesenchymal area. The remnants of the decidua form the placental septa; these septa separate incompletely forming placental cotyledons. In between 15-30 cotyledons are present and the septa restrict the exchange of blood between these cotyledons. [Varney et al. 2004]

Spiral artery remodelling:

During pregnancy the uterine spiral arteries are remodelled into dilated uteroplacental vessels by the mechanism classified as physiological changes [Fig.5]. Spiral arteries develop under the influence of progesterone. After 30-40 days of ovulation the trophoblast starts to cause the erosion of maternal spiral arteries. The remodelling of spiral arteries is very essential for foetal growth and development and supply nutrients to the foetus therefore; they are highly remodelled by invasive trophoblast for this purpose. Spiral arteries originate from the radial arteries that are present at the endometrial or myometrial borders.

Fig.5 showing unmodified & Trophoblast modified artery [cited in Hills 2010]

The trophoblast invades the spiral arteries and leads to the remodelling of these arteries into dilated and inelastic vessels. For this process to occur, these arteries undergo to replace their endothelium and smooth muscle cells, they lose their elasticity, become dilated and there is loss of vasomotor control [Fig.6]. Trophoblast-dependent apoptopic mechanism and cell-cell interactions contribute to the smooth muscle cell loss during remodelling of arteries. Spiral artery remodelling decrease the resistance of maternal blood flow and increase the blood flow towards the placenta to maintain the foetal requirement. The changes that occur during remodelling of arteries can be divided into three steps: - [Kaufmann et al 2003]

1.      Vascular changes that involve trophoblast invasion independent mechanisms that are thought to be mediated by decidual artery renin-angiotensin systems. Initially these arteries undergo alterations in their properties, muscular hypertrophy, vacuolation and dilatation of their lumen.

2.      Remodelling of arteries involving removal of vascular smooth cells and endothelial cells by invasive extravillous trophoblast (EVT).

3.      Infiltration of the artery walls by the endovascular trophoblast by which endothelial cells are replaced with endovascular EVT and extracellular fibrinoid is deposited.

Fig.6 showing various steps in uterine spiral artery remodelling, starting from the non-pregnant stage [cited in Pijnenborg et al. 2006]

Some vascular changes occur before trophoblast invasion but, invasion by trophoblasts increases the remodelling of spiral arteries. The failure of vessels to remodel can cause IUGR, pre-eclampsia or miscarriage. [Pijnenborg et al. 2006]

Pathways of endovascular trophoblast invasion:

The mechanism of trophoblast invasion of uteroplacental arteries is not understood fully. Invasion by endovascular trophoblasts does not occur uniformly at placental bed as invasion being more distinct in the central region. Anatomically endovascular trophoblast has been thought to follow two different types of pathways viz. extravasation or intravasation pathway [Fig.7] [Kaufmann et al 2003]. The theory of extravasation says that endovascular trophoblast cells enter the arterial lumen from the intervillous space by migrating against the blood flow. The trophoblast cells adhere to the endothelium and infiltrate the vessel walls and cause changes in the vascular media leading to the loss of smooth muscle cells and elastic fibres by forming intraluminal trophoblastic plugs. The theory of extravasation is much supported by studies done on rhesus monkey [Blankenship et al 1993]. According to intravasation model there is increased movement into the vessel of interstitial trophoblast from outside and endovascular trophoblasts represent an end stage of differentiation of interstitial trophoblasts. Later, the cells of extravillous trophoblast invade the arterial walls and enter the lumen of spiral arteries. According to Craven et al the peripheral villi are directed by the uteroplacental flow into marginal veins. These peripheral villi adhere to the endothelial surfaces cell columns and the cells of this column extravasate venous walls.

 

Fig.7 showing A- extravasation & B- intravasation [cited in Kaufmann et al. 2003]

Factors influencing trophoblast invasion & spiral artery remodelling:

Various factors [Fig.8] regulate trophblast invasion and remodelling of spiral arteries like HGF (Hepatocyte growth factor) which promotes trophoblast migration & invasion and effects cell mobility of extravillous trophoblast by stimulating an increase in nitric oxide production through the MAPK (Mitogen activated protein kinase) pathway. Members of IGF (Insulin like growth factor) family like IGF-II bind directly to IGF-R2 (Type-2 IGF receptor) and acts through MAPK pathway to induce trophoblast invasion. IGFBP-1 (Insulin like growth factor binding protein-1) is also expressed along with IGF-II. Both IGFBP and IGF-II affect trophoblast invasion independent of each other. TGF-β (Transforming growth factor β) family which consists of TGF-β 1, 2 and 3 are thought to restrict extravillous trophoblast differentiation along the invasive pathway by their anti-proliferative and anti-invasive properties. TGF-β 2 causes up regulation of α₁, α₂, α₃, α₄ integrin expression which leads to increased adhesiveness.. The effects of TGF-β are mediated through the factor H1F1α (Hypoxia inducible factor 1) [Lala & Chakraborty 2003]. LIF (leukaemia inducible factor) which is a member of interleukin-6 is important for spiral artery remodelling.

Apart from this, cell adhesion molecules like integrins, cadherins especially E-cadherin and VE (Vascular endothelial) cadherin, nectin, connexins, trophinin, PECAM-1 (Platelet endothelial cell adhesion molecule) also play an important role in trophoblast differentiation and cell-cell and cell-extracellular matrix interactions [Aplin et al. 2009]. TNF-α (Tumour necrosis factor) can induce trophoblast apoptosis alone or with IFN-γ (Interferon γ). MMP’s (Matrix metalloproteinases) are essential for spiral artery remodelling and angiogenesis. MMP-2 and MMP-9 or gelatinases A and B play a crucial role by causing trophoblast degradation and remodelling of extracellular matrix. MMP-26 which is recently identified is also thought to be involved in tissue remodelling [Cohen et al. 2005]. Many Growth factors and cytokines such as inhibins and activins e.g. activin-A also play pivotal role in decidualization, angiogenesis, implantation and apoptosis [Jones et al 2002]. uNK (uterine natural killer) cells secrete soluble factors that lead to the disruption of smooth muscle cell disruption and PPARγ (Peroxisome proliferator activated receptor-γ) expressed in villous and extravillous cytotrophoblast and syncytiotrophoblast is also one of factors effecting trophoblast differentiation and remodelling of arteries [Fournier et al. 2007].

Fig.8 showing summary of important factors regulating trophoblast invasion & spiral artery remodelling [cited in Hills 2010]

Conclusion:

Differentiation of trophoblast, invasion of extravillous cytotrophoblast into maternal endometrium and spiral artery remodelling during placental development is helpful in establishing an adequate balance between maternal and foetal systems. uNK cells, macrophages and apoptosis and whole range of signalling cascade is also involved which ensure normal pregnancy any deviation can cause early pregnancy loss, pre-eclampsia and IUGR. There are various processes describing about trophoblast invasion and remodelling like trophoblast independent changes, flow interruption by endovascular plugging, the two-wave hypothesis of decidual and myometrial invasion, Intramural incorporation, intravasation & extravasation, combination of both, endovascular mimicry controversy & maternal vascular repair and the role of uNK cells in spiral artery remodelling but, which one holds best is doubtful as each model is deficient in explaining every aspect of invasion and modelling  in one or the other way. I am in favour of a combination of intravasation and extravasation model proposed by Kam et al. along with a role of uNK cells as it justifies infiltration and replacement of arterial media and adventitia by the interstitial trophoblast cells and replacement of endothelium by invading trophoblast afterwards. Furthermore, most of these studies are carried out on human samples so this model sounds more accurate to me as far as its relevance in humans is considered but, it is difficult to draw a perfect conclusion from the data available as research work has its own limitations like poor availability of research tools as the easily available placenta does not cover the required field which is placental bed and the hysterctomized uteri that are available are mostly diseased so it becomes very difficult to get the desired results. Moreover, human samples involve an ethical issue also. The easily available samples are obviously the animal samples but, their comparability to humans is an important question. But, as human placenta is heamochorial which is also found in rats, mice etc. therefore, animal samples can be useful. The development of human trophoblast cell culture has been very useful in discovering information on trophoblast differentiation. Though lot of work is done in this field but, how molecular factors play a role and what mechanism controls trophoblast invasion exactly is not understood fully. Though immense research is done in understanding placental development as a whole still lot of work is required in this area as process of trophoblast invasion and spiral artery remodelling remains quite controversial. So, I think more of research work is required in coming times to understand all of this properly.

         

                                                             Dr.Bharati Sood

Testosterone Synthesis and Signalling

      

Introduction:

Testosterone is an important and main androgen i.e. male sex steroid hormone which plays an essential role in the development, differentiation and functioning of the male reproductive system. Mostly the genomic effects of testosterone are described which require the mediation of androgen receptors. Non-genomic effects of testosterone have been described recently. It is having a basic structure as shown below in Fig.1-

Fig.1 Structure of testosterone (cited in steroid chemistry, 2009)

As testosterone is the principal androgen which supports spermatogenesis therefore, its synthesis is quite important as it influences the development and behaviour patterns in male (Johnson &Everitt 1980). Any deviations from normal testosterone synthesis and signalling that has been described may lead to some serious diseases and problems as such, therefore it is very important to understand how testosterone is synthesized and signalled in the body.

Synthesis of Testosterone:

Testosterone can be synthesized directly within the testis by the leydig cells and also via the inter-conversion from adrenal androgens. (Johnson&Everitt 1980)

Synthesis within the testis:

Like other steroid hormones, testosterone is also derived from common precursor cholesterol. The first step in the synthesis of testosterone is the conversion of cholesterol to pregnenolone. This is the slowest step that requires a large amount of energy for initiation. The conversion of cholesterol to pregnenolone takes place in the inner mitochondria and NADPH, oxygen and cytochrome P-450 are required along with the side chain cleavage reaction of cholesterol (Johnson &Everitt). After cholesterol is converted to pregnenolone, inter conversion takes place in smooth endoplasmic reticulum by a ‘biosynthetic unit’ which consists of a series of enzymes which are arranged together. For testosterone synthesis, this pregnenolone is converted to progesterone in the presence of 3β-dehydrogenase.The formed progesterone is then converted to 17-hydroxyprogesterone by 17-hydroxylase. Further 17-hydroxyprogesterone is converted into androstenedione by 17-hydroxylase. Now androstenedione is converted to testosterone by the action of 17β-hydroxysteroid dehydrogenase (Fig.2) (Hills 2010).

Fig.2 Testicular biosynthetic pathway of testosterone (cited in endotext.org 2009)

Synthesis via inter-conversion from adrenals:

Adrenal cortex is an important place where the production of androgen occurs. Functionally and histologically it can be divided into 3 parts viz. zonaglomerulosa (outer part), zonafasciculata (intermediate part) & the zonareticularis (central part) as shown in Fig.3. (Sanderson 2006)

Fig.3 Cross sections of Adrenal gland (cited in mcg.edu 2008)

Zonareticularis is the principle site where androgen synthesis takes place. The production of testosterone in the adrenals is under the control of Adrenocorticotropic hormone (ACTH). The production of testosterone is increased via the cAMP PK A (cyclic adenosine mono phosphate protein kinase A) pathway. After the conversion of cholesterol to pregnenolone by the action of 17α- hydroxylase, progesterone is hydyroxylated to 17-hydroxypregnenolone by 17α-hydroxylase. This 17-hydroxypregnenolone is further converted to dehydroepiandrosterone(DHEA)  by the action of 17, 20 desmolase. Now, in the zonareticularis this DHEA is converted to androstenedione in the presence of 3β-dehydrogenase. This androstenedione gets converted to testosterone by the action of 17β-hydroxysteroiddehydrogenase.Dehydroepiandrosterone (DHEA) and androstenedione are the adrenal androgens.Testosterone is further converted to dihydrotestosterone (DHT) by 5α-reductase in the testis (Fig.4). In a normal adult man about 6mg of testosterone is produced daily, out of which only 25 µg approximately is stored in the testis, which indicates that testosterone is continuously synthesised and released in the circulation. (Kaplan &Pesce 1984)

Fig.4 Testosterone synthesis pathway (cited in AACR 2010)

Regulation of testosterone expression:

Testosterone synthesis is under the control of hypothalamic-pituitary-gonadal axis. Gonadotropin releasing hormone (GnRH) is produced in the hypothalamus in a pulsatile mode and binds to the specific receptors present on the plasma membrane. At anterior pituitary GnRH stimulation leads to the pulsatile secretions of  luteinizing hormone (LH) and Follicle stimulating hormone (FSH). Testosterone is synthesised by the interstitial cells of leydig of testis by the action of LH. GnRH stimulates LH and it binds to the LH receptors and conversion of cholesterol to testosterone takes place in the interstitial cells of Leydig. LH controls the rate of testosterone synthesis and FSH is required for maintaining spermatogenesis and it acts on sertoli cells. Under the influence of FSH sertoli cells secrete androgen binding protein (ABP) as shown in Fig.5. Testosterone binds with ABP which carries it to seminiferous tubules to facilitate spermatogenesis. As sertoli cells have 5α-reductase, testosterone is converted to a more potent form dihydrotestosterone (DHT) there. (Johnson &Everitt 1980; Kaplan &Pesce 1984)

Fig.5 Regulation of gonadal steroid expression (cited in Hills 2010)

Negative feedback:

The aim of negative feedback is to maintain Homeostasis. The synthesis of testosterone depends on the action of LH and the secretion of LH is itself controlled through the negative feedback of testosterone to hypothalamus. When the amount of testosterone produced is increased, it leads to the suppression of GnRHwhich in turn effects the secretion of LH by pituitary and thus testosterone synthesis is kept under control (Fig.6). For this negative feedback to occur there are some factors which either stop GnRH secretion or inhibit testosterone receptor synthesis and in their absence, the production of testosterone will stop automatically. Thus testosterone regulates the secretions of LH by reducing LH peaks and LH frequency. Certain hormones like inhibin which is produced in sertoli cells, also has a negative feedback on pituitary FSH secretion though the effect is not much. Testosterone can cause the inhibition of both LH and FSH whereas inhibin inhibits the secretions of FSH mainly. (Elder & Dale 2000)

Fig.6 Negative feedback due to increased testosterone (cited in Hills 2010)

Testosterone signalling:

Classical Pathway:

Testosterone is an androgen and to mediate its actions or in order to execute its biological effects, it binds to the intracellular androgen receptor (AR). Androgen receptors are basically proteins which consist of a binding site for a specific androgen. Androgen receptor belongs to the nuclear receptor superfamily and consists of 4 functional domains viz. ligand binding domain (LBD), a DNA binding domain (DBD), a hinge region and an N-terminal domain (NTD). Through the action of LBD, testosterone is able to control the AR activity (Fig.7). DBD recognizes a specific sequence of DNA and the hinge region connects the LBD with DBD (Claessens et. al 2008)

Fig.7 Various domains of androgen receptor (cited in Hills 2010)

Synthesis of testosterone takes place by the classical action of AR. Androgen receptor (AR) has 2 native ligands- testosterone and 5α-DHT. The cytoplasm and nucleus consists of specific AR’s. Testosterone has a great affinity for the AR, therefore, as soon as it comes in contact with the AR; it readily binds to the ARwhich further leads to the conformational changes and this is termed as the activation of AR (Fig.8).

Fig.8 The classical testosterone signalling pathway (Walker 2010)

For the expression of genes, the activated AR binds to the androgen response elements (ARE’s) which are specific DNA sequences. For androgen the consensus DNA (Deoxyribonucleic acid) sequence reads 5'-TGTTCT-3'. In androgen responsive genes, these types of binding sequences have been described. The protein expression is controlled by the promoter of a gene and this is the region where these Androgen response elements (ARE’s) are located. When testosterone is converted to dihydrotestosterone (DHT) by 5α-reductase, the formed DHT binds with the AR and dimerization of AR takes place and this is termed as the activation of AR. This activated receptor has great affinity for the promoter region of DNA which codes for the gene expression. It now enters the nucleus and binds with the ARE’s on the DNA and thus gene expression is promoted (Fig.9). Along with ARE’s there are some other co-activator proteins and transcription factors which bring about the gene expression. Only leydig cells, peritubular cells and sertoli cells in the testis are able to express the AR. Germ cells of the mature testis are not able to express AR. Mature leydig cells have a very good expression for AR. (Walker & Cheng 2005)

Fig.9 Activation of AR and expression of genes (Hills 2010)

Therefore, AR plays a very crucial role in the classical pathway of testosterone signalling. Apart from its role in testosterone signalling, AR performs some other functions as well. From the result of AR knock out and knock-in mouse modelsit was found that activity of AR plays a crucial role in spermatogenesis as follows-:  (Kerkhofs et.al 2009)

1.      AR is necessary for progression through Meiosis I.

2.      During spermatogenesis the round spermatids are converted into elongated ones, which also require the activity of AR.

3.      During the terminal stages of spermatogenesis, AR plays a crucial role.

Non-Classical Pathway:

Apart from the classical mechanism of testosterone signalling, non-classical pathway of testosterone signalling also exists. Various studies of the past prove that testosterone can directly lead to the gene expression without AR binding to DNA. It was first indicated by Fix et al. For these studies, sertoli cells from rat testis were isolated and then cultured. Then they were stimulated with the testosterone levels less than or similar to that found in testis. This led to the increased phosphorylation of the ERK/MAP (extracellular signal-regulated kinase/mitogen activated protein) kinase and the CREB (cAMPresponse element-binding) transcription factor. As the phosphorylation of the CREB and ERK occurred more rapidly than 30-45 minutes.Testosterone also causes activation of kinase p90^RSK (p90 ribosomal S6 kinase) which induces CREB-mediated transcription. All this indicates that some other pathway does exist other than the classical one as this phosphorylation doesn’t take place by the classical mechanism. Apart from this it was found that testosterone plays an important role on the effect on the concentration of intracellular Ca⁺² concentrations. For spermatogenesis testosterone level in sertoli cells should be more than 70nM but, the binding of testosterone to the AR and the gene expression are saturated at 1nM which point towards some other signalling pathway present apart from the classical one. (Walker 2009)

Furthermore, as soon as testosterone stimulation takes place, the Ca⁺² levels in freshly isolated cultures of sertoli cells are elevated, which suggests that testosterone signalling is not fully dependent on the AR and its interactions with the DNA leading to gene expression. If it would have been so then Ca⁺² levels should not be elevated within seconds of elevation of testosterone.In sertoli cells testosterone does not increase the levels of cAMP (cyclic adenosine mono phosphate) but in the activation of MAP (Mitogen activated protein) kinase pathway, CREB (cAMP response element-binding) phosphorylation and CREB mediated transcription, it plays an important role (Fig.10). Two pathways were described in support of this – (Walker & Cheng 2005)

1^st pathway –

Testosterone binds to AR which causes activation of Src (family of protein kinases) and as soon as this activation occurs it leads to the activation of MAP kinase pathway by a series of events by Ras (Rat sarcoma) G-protein (it is a signal transducing guanine nucleotide binding protein).

2^nd pathway –

When testosterone binds to AR there is an increase in the Ca+2 levels. Once this occurs it causes activation of all the intermediates that have the capability of RasorRas like G-protein stimulation which further activates MAP kinase pathway.

Moreover it was found that testosterone can directly activate non-genomic signalling pathways irrespective of AR and DNA interaction. AR mediates testosterone induced MAP kinase and CREB (cAMP response element-binding) phosphorylation. Following evidence supports it – (Walker & Cheng 2005)

1^st – Flutamide which is an AR antagonist leads to the inhibition of testosterone mediated CREB phosphorylation.

2^nd – Sertoli cells which did not have the activity of AR, CREB phosphorylation did not take place in them.

3^rd – Sertoli cells which had the testicular feminization (tfm) AR mutant, testosterone could not increase the CREB phosphorylation in them.

Fig.10 Non-classical testosterone signalling pathways (Walker 2010)

All of these series of evidences (Walker & Cheng 2005; Walker 2009; Walker 2010) suggest that apart from the classical mechanism, testosterone acts through other pathways also in the sertoli cells. Both classical and non-classical pathways (Fig.11) play an important role in spermatogenesis and maintainingfertility.In the future, may be it will be proved that there is some co-relation between these two pathways or they may be interdependent. It may give a breakthrough in determining the male fertility as well. As most of the studies are being conducted on ratsand mice therefore, their relevance to human beings still remains a question.But, as the reproductive system of humans and rats is very much identical therefore, studies on rats remain a good source of research and information. In vivo studies will be more useful in future.

Fig.11 Classical & non-classical testosterone pathways (Walker 2009)

Conclusion:

A lot of research work is done on understanding the role of testosterone in body, its functioning, synthesis, signalling and its role in spermatogenesis. Apart from this the action of AR, AR antagonists, hormonal control of testosterone, and negative feedback by testosterone, gene expression and Ca⁺² uptakes by sertolicells is well understood today. Some years back testosterone was thought to act only through the classical mechanism but, recent research and evidences prove that apart from the classical pathway, a non-classical pathway which includes CREB and MAP kinase pathway also exists. Non-classical pathway of testosterone signalling is not yet understood fully; still the role of hormones and co-regulators remains to be fully described and most of the study has been conducted in vitro, therefore, in vivo study can provide some useful information. So, may be in coming years there will be more research in this area which will provide more information about testosterone signalling and also about the co-factors which are yet to be identified.

Dr.Bharati Sood