Rex A. Hess

A role for oestrogens in the male reproductive system

Oestrogen is considered to be the ‘female’ hormone, whereas testosterone is considered the ‘male’ hormone. However, both hormones are present in both sexes. Thus sexual distinctions are not qualitative differences, but rather result from quantitative divergence in hormone concentrations and differential expressions of steroid hormone receptors. In males, oestrogen is present in low concentrations in blood, but can be extraordinarily high in semen, and as high as 250 pg ml−1in rete testis fluids,, which is higher than serum oestradiol in the female. It is well known that male reproductive tissues express oestrogen receptors, but the role of oestrogen in male reproduction has remained unclear. Here we provide evidence of a physiological role for oestrogen in male reproductive organs. We show that oestrogen regulates the reabsorption of luminal fluid in the head of the epididymis. Disruption of this essential function causes sperm to enter the epididymis diluted, rather than concentrated, resulting in infertility. This finding raises further concern over the potential direct effects of environmental oestrogens on male reproduction and reported declines in human sperm counts,.

Estrogen in the adult male reproductive tract: A review

Testosterone and estrogen are no longer considered male only and female only hormones. Both hormones are important in both sexes. It was known as early as the 1930s that developmental exposure to a high dose of estrogen causes malformation of the male reproductive tract, but the early formative years of reproductive biology as a discipline did not recognize the importance of estrogen in regulating the normal function of the adult male reproductive tract. In the adult testis, estrogen is synthesized by Leydig cells and the germ cells, producing a relatively high concentration in rete testis fluid. Estrogen receptors are present in the testis, efferent ductules and epididymis of most species. However, estrogen receptor-α is reported absent in the testis of a few species, including man. Estrogen receptors are abundant in the efferent ductule epithelium, where their primary function is to regulate the expression of proteins involved in fluid reabsorption. Disruption of the α-receptor, either in the knockout (αERKO) or by treatment with a pure antiestrogen, results in dilution of cauda epididymal sperm, disruption of sperm morphology, inhibition of sodium transport and subsequent water reabsorption, increased secretion of Cl-, and eventual decreased fertility. In addition to this primary regulation of luminal fluid and ion transport, estrogen is also responsible for maintaining a differentiated epithelial morphology. Thus, we conclude that estrogen or its α-receptor is an absolute necessity for fertility in the male.

ERM is required for transcriptional control of the spermatogonial stem cell niche

Division of spermatogonial stem cells produces daughter cells that either maintain their stem cell identity or undergo differentiation to form mature sperm. The Sertoli cell, the only somatic cell within seminiferous tubules, provides the stem cell niche through physical support and expression of surface proteins and soluble factors. Here we show that the Ets related molecule (ERM) is expressed exclusively within Sertoli cells in the testis and is required for spermatogonial stem cell self-renewal. Mice with targeted disruption of ERM have a loss of maintenance of spermatogonial stem cell self-renewal without a block in normal spermatogenic differentiation and thus have progressive germ-cell depletion and a Sertoli-cell-only syndrome. Microarray analysis of primary Sertoli cells from ERM-deficient mice showed alterations in secreted factors known to regulate the haematopoietic stem cell niche. These results identify a new function for the Ets family transcription factors in spermatogenesis and provide an example of transcriptional control of a vertebrate stem cell niche.

Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen

We report the immortalization, using the SV40 large T antigen, of all the cell types contributing to a developing seminiferous tubule in the mouse testis. Sixteen peritubular, 22 Leydig, 8 Sertoli, and 1 germ cell line have been established and cultured successfully for 90 generations in a period of 2.5 years. Immortalized peritubular cells were identified by their spindle-like appearance, their high expression of alkaline phosphatase, and their expression of the intermediary filament desmin. They also produce high amounts of collagen. Immortalized Leydig cells are easily identifiable by the accumulation of lipid droplets in their cytoplasm and the production of the enzyme 3-beta-hydroxysteroid dehydrogenase. Some Leydig cell lines also express LH receptors. The immortalized Sertoli cells are able to adopt their typical in vivo columnar appearance when cultured at high density. They exhibit a typical indented nucleus and cytoplasmic phagosomes. Some Sertoli cell lines also express FSH receptors. A germ cell line (GC-1spg) was established that corresponds to a stage between spermatogonia type B and primary spermatocyte, based on its characteristics in phase contrast and electron microscopy. This cell line expresses the testicular cytochrome ct and lactate dehydrogenase-C4 isozyme. These four immortalized cell types, when plated together, are able to reaggregate and form structures resembling two-dimensional spermatogenic tubules in vitro. When only the immortalized somatic cells are cocultured, the peritubular and Sertoli cells form cord-like structures in the presence of Leydig cells. Fresh pachytene spermatocytes cocultured with the immortalized somatic cells integrate within the cords and are able to survive for at least 7 days. The ability to perform coculture experiments with immortalized testicular cell lines represents an important advancement in our ability to study the nature of cell-cell and cell-matrix interactions during spermatogenesis and testis morphogenesis.

Oestrogens and spermatogenesis

The role of oestrogens in male reproductive tract physiology has for a long time been a subject of debate. The testis produces significant amounts of oestrogenic hormones, via aromatase, and oestrogen receptors (ERs)α (ESR1) and ERβ (ESR2) are selectively expressed in cells of the testis as well as the epididymal epithelium, depending upon species. This review summarizes the current knowledge concerning the presence and activity of aromatase and ERs in testis and sperm and the potential roles that oestrogens may have in mammalian spermatogenesis. Data show that physiology of the male gonad is in part under the control of a balance of androgens and oestrogens, with aromatase serving as a modulator.

Estrogen action and male fertility: Roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function

Estrogen receptor α (ERα) is essential for male fertility. Its activity is responsible for maintaining epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. These discoveries and others have helped to establish estrogens bisexual role in reproductive importance. Reported here is the molecular mechanism to explain estrogens role in fluid reabsorption in the male reproductive tract. It is shown that estrogen regulates expression of the Na+/H+ exchanger-3 (NHE3) and the rate of 22Na+ transport, sensitive to an NHE3 inhibitor. Immunohistochemical staining for NHE3, carbonic anhydrase II (CAII), and aquaporin-I (AQP1) was decreased in ERα knockout (αERKO) efferent ductules. Targeted gene-deficient mice were compared with αERKO, and the NHE3 knockout and CAII-deficient mice showed αERKO-like fluid accumulation, but only the NHE3 knockout and αERKO mice were infertile. Northern blot analysis showed decreases in mRNA for NHE3 in αERKO and antiestrogen-treated mice. The changes in AQP1 and CAII in αERKO seemed to be secondary because of the disruption of apical cytoarchitecture. Ductal epithelial ultrastructure was abnormal only in αERKO mice. Thus, in the male, estrogen regulates one of the most important epithelial ion transporters and maintains epithelial morphological differentiation in efferent ductules of the male, independent of its regulation of Na+ transport. Finally, these data raise the possibility of targeting ERα in developing a contraceptive for the male.

Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) in Spermatogenic Cells Induced by Retinoic Acid: An In Vivo Study in Vitamin A-Sufficient Postnatal Murine Testes

Abstract Vitamin A is required for male fertility and normal spermatogenesis. Retinoic acid (RA), an active metabolite of vitamin A, is necessary for spermatogonial maturation and proper entry of germ cells into meiotic prophase in the postnatal testes. The expression of Stra8, which is essential for successful meiosis in both male and female gonads and normal spermatogenesis, is directly related to the availability of RA. This study examined the developmental expression pattern of Stra8 transcript in both male and female gonads, provided specific cellular localization of STRA8 protein in the postnatal and adult testis, and investigated RA actions in adult germ cells in a vitamin A-sufficient condition. The peak of Stra8 mRNA expression coincided with the onset of meiosis in postnatal testes. STRA8 protein was detected in gonocytes as early as 5 days postpartum. The expression of STRA8 protein in the neonatal testes was not uniform among spermatogonia, perhaps heralding the asynchronous beginning of spermatogenesis. In adult testes, the highest level of Stra8 mRNA and protein was found in seminiferous epithelial stages VI–VIII. STRA8 protein was localized to some type A and B spermatogonia, preleptotene spermatocytes, and early leptotene spermatocytes. In the vitamin A-sufficient adult testes, RA but not retinol acetate stimulated Stra8 mRNA expression. STRA8 protein expression in adult spermatogonia was induced by RA stimulation, suggesting its role in spermatogonial differentiation. Retinoic acid also increased the number of preleptotene spermatocytes exhibiting 5-bromo-2-deoxyuridine incorporation, indicating a more synchronized premeiotic DNA replication.

Estrogen Receptor α Has a Functional Role in the Mouse Rete Testis and Efferent Ductules

Abstract Previous studies of the estrogen receptor-alpha knockout (αERKO) in the male mouse demonstrate that the rete testis and efferent ductules are targets of estrogen. Because the αERKO mouse lacks a functional estrogen receptor alpha (ERα) throughout development, it was not known whether the morphological and physiological abnormalities observed in the αERKO male were due to developmental defects or to dysfunctions concurrent with the lack of ERα in the tissue. This study was designed to determine if treatment of normal wild-type (WT) mice with the pure antiestrogen, ICI 182,780, (ICI) could reproduce the morphological characteristics seen in αERKO mice. Thirty-day-old male mice were treated for 35 days with either castor oil or ICI. Age-equivalent αERKO mice were used for comparison. Light microscopic examinations of the reproductive tracts revealed dramatic changes in the efferent ductules of treated mice: a 1.7-fold increase in luminal diameter, a 56% reduction in epithelial cell height, a 60% reduction in brush boarder height of nonciliated cells, and an apparent reduction of the number of observable lysosomes and endocytotic vesicles. Testes of ICI-treated mice showed swollen rete testes area (6.5 times larger than control) and a 65% reduction in rete testis epithelium height. However, there were no significant changes in body and testis weights. These results indicate that ER blockage with ICI in WT mice results in morphological changes of the efferent ductules resembling those seen in αERKO siblings of the same age. Based on this study, we conclude that ERα has a functional role in the mouse reproductive tract and the aberrant morphology observed in the efferent ductules of the αERKO mouse is likely the result of a concurrent response to the lack of functional ERα, and not solely due to the lack of ERα during early developmental times.

Oestrogen, its receptors and function in the male reproductive tract - a review.

Oestrogen is synthesized in the male reproductive system by at least three different cell types; Sertoli, Leydig and germ cells. Although testosterone is recognized as the primary sex steroid in man, oestrogen is produced in sizable quantities in the testis, as well as the brain and is found in extremely high concentrations in the semen of several species. The high concentration of oestrogen in rete testis fluid of the rodent is now thought to be derived from the conversion of testosterone to estradiol by P450 aromatase in germ cells of the testis and spermatozoa traversing the reproductive tract. This new major source of oestrogen would target oestrogen receptors in the male reproductive tract, in particular the efferent ductules, which contain the highest concentration of oestrogen receptor-alpha. This recent data raises new hypotheses regarding the role of oestrogen in the function of the male reproductive system. The oestrogen receptor-alpha knockout mouse was used to help define the function of oestrogen in the male. It was found that oestrogen receptor-alpha is essential for fluid reabsorption in the efferent ductules and in the absence of expression the male is infertile.

Differential Expression of Estrogen Receptors α and β in the Reproductive Tractsof Adult Male Dogs and Cats

Abstract Expression of estrogen receptors (ERs) in the reproductive tracts of adult male dogs and cats has not been reported. In the present study, ERα and ERβ were localized by immunohistochemistry using ER-specific antibodies. ERα was found in interstitial cells and peritubular myoid cells in the dog testis, but only in interstitial cells of the cat. In rete testis of the dog, epithelial cells were positive for ERα staining, but in the cat, rete testis epithelium was only weakly positive. In efferent ductules of the dog, both ciliated and nonciliated cells stained intensely positive. In the cat, ciliated epithelial cells were less stained than nonciliated epithelial cells. Epithelial cells in dog epididymis and vas deferens were negative for ERα. In the cat, except for the initial region of caput epididymis, ERα staining was positive in the epithelial cells of epididymis and vas deferens. Multiple cell types of dog and cat testes stained positive for ERβ. In rete testis and efferent ductules, epithelial cells were weakly positive for ERβ. Most epithelial cells of the epididymis and vas deferens exhibited a strong positive staining in both species. In addition, double staining was used to demonstrate colocalization of both ERα and ERβ in efferent ductules of both species. The specificity of antibodies was demonstrated by Western blot analysis. This study reveals a differential localization of ERα and ERβ in male dog and cat reproductive tracts, demonstrating more intensive expression of ERβ than ERα. However, as in other species, the efferent ductules remained the region of highest concentration of ERα.