Peter J. O'Shaughnessy

Changes in Leydig Cell Gene Expression During Development in the Mouse

Abstract Developmental changes in the expression of 18 Leydig cell-specific mRNA species were measured by real-time polymerase chain reaction to partially characterize the developmental phenotype of the cells in the mouse and to identify markers of adult Leydig cell differentiation. Testicular interstitial webs were isolated from mice between birth and adulthood. Five developmental patterns of gene expression were observed. Group 1 contained mRNA species encoding P450 side chain cleavage (P450scc), P450c17, relaxin-like factor (RLF), glutathione S-transferase 5-5 (GST5-5), StAR protein, LH receptor, and epoxide hydrolase (EH); group 2 contained 3β-hydroxysteroid dehydrogenase (3β-HSD) VI, 17β-hydroxysteroid dehydrogenase (17β-HSD) III, vascular cell adhesion molecule 1, estrogen sulfotransferase, and prostaglandin D (PGD)-synthetase; group 3 contained patched and thrombospondin 2 (TSP2); group 4 contained 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase; group 5 contained sulfonylurea receptor 2 and 3β-HSD I. Group 1 contained genes that were expressed in fetal and adult Leydig cells and which increased in expression around puberty toward a maximum in the adult. Group 2 contained genes expressed only in the adult Leydig cell population. Group 3 contained genes with predominant fetal/neonatal expression in the interstitial tissue. Group 4 contained genes with a peak of expression around puberty, whereas genes in group 5 show little developmental change in expression. Highest mRNA levels in descending order were RLF, P450c17, EH, 17β-HSD III, PGD-synthetase, GST5-5, and P450scc. Results identify five genes expressed in the mouse adult Leydig cell population, but not in the fetal population, and one gene (TSP2) that may be expressed only in the fetal Leydig cell population. The developmental pattern of gene expression suggests that three distinct phases of adult Leydig cell differentiation occur.

Hormonal control of germ cell development and spermatogenesis

Spermatogenesis is completely dependent on the pituitary hormone follicle-stimulating hormone (FSH) and androgens locally produced in response to luteinising hormone (LH). This dual control has been known since the 1930s and 1940s but more recent work, particularly using transgenic mice, has allowed us to determine which parts of the spermatogenic pathway are regulated by each hormone. During the first spermatogenic cycle after puberty both FSH and androgen act to limit the massive wave of germ cell apoptosis which occurs at this time. The established role of FSH in all cycles is to increase spermatogonial and subsequent spermatocyte numbers with a likely effect also on spermiation. Mice lacking FSH or its receptor are fertile, albeit with reduced germ cell numbers, and so this hormone is not an essential regulator of spermatogenesis but acts to optimise germ cell production Androgens also appear to regulate spermatogonial proliferation but, crucially, they are also required to allow spermatocytes to complete meiosis and form spermatids. Animals lacking androgen receptors fail to generate post-meiotic germ cells, therefore, and are infertile. There is also strong evidence that androgens act to ensure appropriate spermiation of mature spermatids. Androgen regulation of spermatogenesis is dependent upon action on the Sertoli cell but recent studies have shown that androgenic stimulation of the peritubular myoid cells is also essential for normal germ cells development. While FSH or androgen alone will both stimulate germ cell development, together they act synergistically to maximise germ cell number. The other hormones/local factors which can regulate spermatogenesis include activins and estrogens although their role in normal physiological regulation of this process needs to be more clearly established. Regulation of spermatogenesis in primates appears to be similar to that in rodents although the role of FSH may be greater. While our knowledge of hormone function during spermatogenesis is now well developed we still lack understanding of the mechanisms by which these hormones act to regulate this process.

Expression of follicle stimulating hormone-receptor mRNA during gonadal development

Receptors for follicle-stimulating hormone (FSH) are found only in the gonads and have been localised to the Sertoli cells of the testis and the granulosa cells of the ovary. During gonadal development, functional signal transduction systems are present before gonadotrophin receptors appear indicating the expression of the receptors is the crucial step in development of gonadal responsiveness to gonadotrophins. The FSH receptor gene contains a single large exon which encodes the transmembrane and intracellular domains and nine smaller exons which encode most of the extracellular domain. In all species studied so far the FSH-receptor primary transcript has been shown to undergo alternate splicing. The function of these alternate transcripts is unclear but changes in alternate splicing appear to be associated with development of receptor mRNA expression. In the rat transcripts encoding only the extracellular domain of the receptor are detectable 2 days before transcripts encoding the full length receptor. In the mouse ovary FSH-receptor mRNA levels and alternate splicing has been measured during development. Results show that FSH-receptor mRNA is detectable in day 1 ovaries which contain only primordial follicles. At this stage mRNA levels are low but a significant increase in FSH-receptor mRNA is seen around day 5 when primary follicles first appear. This correlates with in situ hybridisation studies which first detect FSH-receptor transcripts in primary follicles. At all stages of development the level of transcripts encoding the extracellular domain was significantly greater than that encoding for the transmembrane and intracellular regions suggesting that significant levels of shortened transcripts are produced. In the hypogonadal (hpg) mouse which lacks circulating gonadotrophins levels of FSH-receptor mRNA appeared normal up to 15 days. This shows that gonadotrophins ar not require for development of FSH-receptor mRNA levels. Studies on FSH-receptor mRNA levels during granulosa cell luteinization show that there is complete loss of full-length transcripts soon after luteinization. Transcripts encoding the extracellular domain remain present, however, up to at least mid-cycle. Thus, changes in receptor transcript splicing during loss of FSH-receptors appear to mimic, in reverse, changes occurring during development. It may be that the FSH-receptor gene is constitutively expressed in follicular (pre-granulosa) cells, granulosa cells and granulosa-luteal cells but that control of RNA splicing regulates levels of full-length FSH-receptor transcript.

Spermatogenesis and Sertoli cell activity in mice lacking Sertoli cell receptors for follicle stimulating hormone and androgen

Spermatogenesis in the adult male depends on the action of FSH and androgen. Ablation of either hormone has deleterious effects on Sertoli cell function and the progression of germ cells through spermatogenesis. In this study we generated mice lacking both FSH receptors (FSHRKO) and androgen receptors on the Sertoli cell (SCARKO) to examine how FSH and androgen combine to regulate Sertoli cell function and spermatogenesis. Sertoli cell number in FSHRKO-SCARKO mice was reduced by about 50% but was not significantly different from FSHRKO mice. In contrast, total germ cell number in FSHRKO-SCARKO mice was reduced to 2% of control mice (and 20% of SCARKO mice) due to a failure to progress beyond early meiosis. Measurement of Sertoli cell-specific transcript levels showed that about a third were independent of hormonal action on the Sertoli cell, whereas others were predominantly androgen dependent or showed redundant control by FSH and androgen. Results show that FSH and androgen act through redundant, additive, and synergistic regulation of spermatogenesis and Sertoli cell activity. In addition, the Sertoli cell retains a significant capacity for activity, which is independent of direct hormonal regulation.

Role of androgen and gonadotrophins in the development and function of the Sertoli cells and Leydig cells: Data from mutant and genetically modified mice

Development and maintenance of the male phenotype and establishment of fertility are all dependent upon the activity of the Sertoli cells and Leydig cells of the testis. This review examines the regulation and function of these cell during fetal and post-natal development. Fetal Leydig cells are sensitive to both luteinising hormone (LH) and adrenocorticotrophic hormone (ACTH) but Leydig cell function appears normal in fetal mice lacking both hormones or their receptors. Post-natally, the Sertoli cells and Leydig cells are reliant upon the pituitary gonadotrophins. Leydig cells are critically dependent on LH but follicle-stimulating hormone (FSH), presumably acting through the Sertoli cell, can also affect Leydig cell function. Testosterone secreted by the Leydig cells acts with FSH to stimulate Sertoli cell activity and spermatogenesis. Study of animals lacking FSH-receptors and androgen-receptors shows that both hormones can act to maintain the meiotic germ cell population but that androgens are critical for completion of meiosis.

Follicle-stimulating hormone receptor mRNA in the mouse ovary during post-natal development in the normal mouse and in the adult hypogonadal (hpg) mouse: structure of alternate transcripts

The structure of RNA encoding the mouse ovarian follicle-stimulating hormone (FSH) receptor was studied during post-natal development and in the adult hypogonadal (hpg) mouse which lacks circulating gonadotrophins. Using reverse transcription and the polymerase chain reaction (PCR) four major transcripts of the FSH receptor were found in the normal adult ovary. The largest transcript was the expected size from the position of the PCR primers (on exons 1 and 10) and sequencing confirmed that it was derived from FSH receptor mRNA. The three other transcripts were also derived from FSH receptor mRNA but they contained deletions corresponding to one or more complete exons. Each transcript lacked exon 2 while exons 5 and/or 6 were lacking in the smaller species. All four transcripts were present in ovaries of hpg mice showing that expression of receptor mRNA and development of alternate splicing are not gonadotrophin-dependent. During development in the mouse full-length FSH receptor transcripts were not detected in the ovary until day 5 although shorter transcripts were present at days 1 and 3. Results confirm that the FSH receptor primary transcript undergoes alternate splicing in the ovary and that the pattern of splicing changes as the ovary develops, probably as a result of follicular development.

Development of steroid signaling pathways during primordial follicle formation in the human fetal ovary.

CONTEXT Ovarian primordial follicle formation is critical for subsequent human female fertility. It is likely that steroid, and especially estrogen, signaling is required for this process, but details of the pathways involved are currently lacking. OBJECTIVE The aim was to identify and characterize key members of the steroid-signaling pathway expressed in the second trimester human fetal ovary. DESIGN We conducted an observational study of the female fetus, quantifying and localizing steroid-signaling pathway members. SETTING The study was conducted at the Universities of Aberdeen, Edinburgh, and Glasgow. PATIENTS/PARTICIPANTS Ovaries were collected from 43 morphologically normal human female fetuses from women undergoing elective termination of second trimester pregnancies. MAIN OUTCOME MEASURES We measured mRNA transcript levels and immunolocalized key steroidogenic enzymes and steroid receptors, including those encoded by ESR2, AR, and CYP19A1. RESULTS Levels of mRNA encoding the steroidogenic apparatus and steroid receptors increased across the second trimester. CYP19A1 transcript increased 4.7-fold during this period with intense immunostaining for CYP19A detected in pregranulosa cells around primordial follicles and somatic cells around oocyte nests. ESR2 was localized primarily to germ cells, but androgen receptor was exclusively expressed in somatic cells. CYP17A1 and HSD3B2 were also localized to oocytes, whereas CYP11A1 was detected in oocytes and some pregranulosa cells. CONCLUSIONS The human fetal ovary expresses the machinery to produce and detect multiple steroid signaling pathways, including estrogenic signaling, with the oocyte acting as a key component. This study provides a step-change in our understanding of local dynamics of steroid hormone signaling during the key period of human primordial follicle formation.

Effect of FSH on testicular morphology and spermatogenesis in gonadotrophin-deficient hypogonadal mice lacking androgen receptors.

FSH and androgen act to stimulate and maintain spermatogenesis. FSH acts directly on the Sertoli cells to stimulate germ cell number and acts indirectly to increase androgen production by the Leydig cells. In order to differentiate between the direct effects of FSH on spermatogenesis and those mediated indirectly through androgen action, we have crossed hypogonadal (hpg) mice, which lack gonadotrophins, with mice lacking androgen receptors (AR) either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with recombinant FSH for 7 days and testicular morphology and cell numbers were assessed. In untreated hpg and hpg.SCARKO mice, germ cell development was limited and did not progress beyond the pachytene stage. In hpg.ARKO mice, testes were smaller with fewer Sertoli cells and germ cells compared to hpg mice. Treatment with FSH had no effect on Sertoli cell number but significantly increased germ cell numbers in all groups. In hpg mice, FSH increased the numbers of spermatogonia and spermatocytes, and induced round spermatid formation. In hpg.SCARKO and hpg.ARKO mice, in contrast, only spermatogonial and spermatocyte numbers were increased with no formation of spermatids. Leydig cell numbers were increased by FSH in hpg and hpg.SCARKO mice but not in hpg.ARKO mice. Results show that in rodents 1) FSH acts to stimulate spermatogenesis through an increase in spermatogonial number and subsequent entry of these cells into meiosis, 2) FSH has no direct effect on the completion of meiosis and 3) FSH effects on Leydig cell number are mediated through interstitial ARs.

Direct Action through the Sertoli Cells Is Essential for Androgen Stimulation of Spermatogenesis

Androgens act to stimulate spermatogenesis through androgen receptors (ARs) on the Sertoli cells and peritubular myoid cells. Specific ablation of the AR in either cell type will cause a severe disruption of spermatogenesis. To determine whether androgens can stimulate spermatogenesis through direct action on the peritubular myoid cells alone or whether action on the Sertoli cells is essential, we crossed hypogonadal (hpg) mice that lack gonadotrophins and intratesticular androgen with mice lacking ARs either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with testosterone (T) or dihydrotestosterone (DHT) for 7 d and testicular morphology and cell numbers assessed. Androgen treatment did not affect Sertoli cell numbers in any animal group. Both T and DHT increased numbers of spermatogonia and spermatocytes in hpg mice, but DHT has no effect on germ cell numbers in hpg.SCARKO and hpg.ARKO mice. T increased germ cell numbers in hpg.SCARKO and hpg.ARKO mice, but this was associated with stimulation of FSH release. Results show that androgen stimulation of spermatogenesis requires direct androgen action on the Sertoli cells.

Effect of germ cell depletion on levels of specific mRNA transcripts in mouse Sertoli cells and Leydig cells

It has been shown that testicular germ cell development is critically dependent upon somatic cell activity but, conversely, the extent to which germ cells normally regulate somatic cell function is less clear. This study was designed, therefore, to examine the effect of germ cell depletion on Sertoli cell and Leydig cell transcript levels. Mice were treated with busulphan to deplete the germ cell population and levels of mRNA transcripts encoding 26 Sertoli cell-specific proteins and 6 Leydig cell proteins were measured by real-time PCR up to 50 days after treatment. Spermatogonia were lost from the testis between 5 and 10 days after treatment, while spermatocytes were depleted after 10 days and spermatids after 20 days. By 30 days after treatment, most tubules were devoid of germ cells. Circulating FSH and intratesticular testosterone were not significantly affected by treatment. Of the 26 Sertoli cell markers tested, 13 showed no change in transcript levels after busulphan treatment, 2 showed decreased levels, 9 showed increased levels and 2 showed a biphasic response. In 60% of cases, changes in transcript levels occurred after the loss of the spermatids. Levels of mRNA transcripts encoding Leydig cell-specific products related to steroidogenesis were unaffected by treatment. Results indicate (1) that germ cells play a major and widespread role in the regulation of Sertoli cell activity, (2) most changes in transcript levels are associated with the loss of spermatids and (3) Leydig cell steroidogenesis is largely unaffected by germ cell ablation.