Cathepsin A is expressed in a cell- and region-specific manner in the testis and epididymis and is not regulated by testicular or pituitary factors.

The epithelial cells of the testis are involved in the production, differentiation, and sustenance of sperm, and those of the epididymis play a major role in sperm maturation, protection, and storage. These tissues express various proteins that respond differently to androgens. Cathepsin A is a multifunctional lysosomal carboxypeptidase that also functions as a protective and an activator protein for neuraminidase and beta-galactosidase. In this study, cathepsin A was immunolocalized by light and electron microscopy using a polyclonal affinity-purified antibody on the testis and epididymis of normal, orchidectomized with or without testosterone supplementation, efferent duct-ligated, and hypophysectomized adult rats. In normal rats, cathepsin A expression was noted in lysosomes of Sertoli and Leydig cells but not in germ cells of the testis, as well as nonciliated cells of the efferent ducts. In the epididymis, a cell- and region-specific distribution of cathepsin A was noted. In experimentally treated animals, no changes were noted in the expression of cathepsin A. Immunolabeling of tissues examined at the electron microscopic level revealed that lysosomes were reactive. These data indicate cell- and region-specific expression of cathepsin A in cells of the testis and epididymis and also indicate that cathepsin A expression is not regulated by testicular or pituitary factors.     

High level of endostatin in epididymal epithelium: protection against primary malignancies in this organ?

Rete testis and epididymis are rare locations for primary tumors or metastasis. Assuming that this may be related to expression level of angiogenic inhibitors, we focused our study on the expression pattern of collagen 18/endostatin. In situ hybridization and immunohistochemistry for collagen 18 and endostatin were carried out on sections of human rete testis and epididymis as well as on epididymal adenoma and human testicular tissue with or without carcinoma in situ (CIS). In situ hybridization revealed strong expression of collagen 18 mRNA in rete testis, efferent ducts and epididymal duct. Immunostaining showed collagen 18 in epithelium and basement membrane as well as in blood vessels of rete testis. Further, in both efferent ducts and epididymal duct, collagen 18 was mainly localized in the basement membrane of these ducts and of the blood vessel wall. Endostatin immunostaining was localized in the epithelium of rete testis, efferent ducts and epididymal duct. This pattern of endostatin staining was absent in epididymal adenoma tissue while tumor associated blood vessels exhibited strong endostatin staining. No endostatin staining was detectable in normal germinal epithelium and CIS cells while Leydig cells exhibited strong endostatin staining. High endostatin expression in epididymis may protect this organ against tumor development. Gene therapeutic strategies providing high expression of endostatin in normal epithelia may be useful to prevent tumor development.     

Expression of constitutively active Notch1 in male genital tracts results in ectopic growth and blockage of efferent ducts, epididymal hyperplasia and sterility

The Notch signaling pathway is involved in a variety of developmental processes. Here, we characterize the phenotypes developing in the reproductive organs of male transgenic (Tg) mice constitutively expressing the activated mouse Notch1 intracellular domain (Notch1(intra)) under the regulatory control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Tg expression was detected in testis, vas deferens and epididymis by Northern blot analysis. In situ hybridization with a Notch1-specific probe lacked sensitivity to detect expression in normal-appearing cells, but demonstrated expression in hyperplastic epithelial cells of the vas deferens, epididymis and efferent ducts. Tg males from three independent founder lines were sterile. Histological analysis of reproductive organs of young Tg males (postnatal ages 8 and 21) showed no difference compared to those of non-Tg males. In contrast, in adult Tg mice from day 38 onwards, the efferent ducts, the vas deferens and most epididymal segments revealed bilateral epithelial cell hyperplasia with absence of fully differentiated epithelial cells. Electron microscopy confirmed the uniformly undifferentiated state of these cells. Immunohistochemistry with anti-PCNA antibody also revealed enhanced proliferation of Tg epididymis. In adult Tg testis, the different generations of germ cells of seminiferous tubules appeared normal, although some tubules were highly dilated and revealed an absence of early and/or late spermatids. The epithelial cells of the Tg tubuli recti and rete testis were not abnormal, but the rete testis was highly dilated and contained numerous spermatozoa, suggesting a downstream blockage. Consistent with a blockage of efferent ducts often seen at the rete testis/efferent duct interface, spermatozoa were absent in epididymis of all adult Tg mice and in all highly hyperplastic efferent duct tubules of these Tg mice. Such a blockage was visualized by injection of Evans blue dye into the rete testis lumen. Finally, the presence of ectopic hyperplastic efferent duct tubules was observed within the testicular parenchyma itself, outside their normal territory, suggesting that Notch1 signaling is involved in the establishment of these borders. This phenotype seems to represent a novel developmental defect in mammals. Together, these results show that constitutive Notch1 signaling significantly affects the development of male reproductive organs.     

Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa.

We have attempted to transfect testicular spermatozoa with plasmid DNA by direct injection into testes to obtain transgenic animals [this technique was thus termed "testis-mediated gene transfer (TMGT)"]. When injected males were mated with superovulated females 2 and 3 days after injection, (i) high efficiencies (more than 50%) of gene transmission were achieved in the mid-gestational F0 fetuses, (ii) the copy number of plasmid DNA in the fetuses was estimated to be less than 1 copy per diploid cell, and (iii) overt gene expression was not found in these fetuses. These findings suggest the possibility that plasmid DNA introduced into a testis is rapidly transported to the epididymis and then incorporated by epididymal spermatozoa. The purpose of this study was to elucidate the mechanism of TMGT by introducing trypan blue (TB) or Hoechst 33342 directly into testis. We found that TB is transported to the ducts of the caput epididymis via rete testis within 1 min after testis injection, and TB reached the corpus and cauda epididymis within 2-4 days after injection. Staining of spermatozoa isolated from any portion of epididymis was observed 4 days after injection of a solution containing Hoechst 33342. Injection of enhanced green fluorescent protein (EGFP) expression vector/liposome complex into testis resulted in transfection of epithelial cells of epididymal ducts facing the lumen, although the transfection efficiency appeared to be low. In vivo electroporation toward the caput epididymis immediately after injection of EGFP expression vector into a testis greatly improved the uptake of foreign DNA by the epididymal epithelial cells. PCR analysis using spermatozoa isolated from corpus and cauda epididymis 4 days after injection of a DNA/liposome complex into testis revealed exogenous DNA in these spermatozoa even after treatment with DNase I. These findings indicate that exogenous DNA introduced into tesits is rapidly transported to epididymal ducts via the rete testis and efferent ducts, and then incorporated by epithelial cells of epididymis and epididymal spermatozoa.     

Morphology and immunolocalization of aquaporins 1 and 9 in the agouti (Dasyprocta azarae) testis excurrent ducts

This study investigated the morphology and immunoexpression of aquaporins (AQPs) 1 and 9 in the rete testis, efferent ducts, epididymis, and vas deferens in the Azara’s agouti (Dasyprocta azarae). For this purpose, ten adult sexually mature animals were used in histologic and immunohistochemical analyses. The Azara’s agouti rete testis was labyrinthine and lined with simple cubic epithelium. Ciliated and non-ciliated cells were observed in the epithelium of the efferent ducts. The epididymal cellular population was composed of principal, basal, apical, clear, narrow, and halo cells. The epithelium lining of vas deferens was composed of the principal and basal cells. AQPs 1 and 9 were not expressed in the rete testis. Positive reaction to AQP1 was observed at the luminal border of non-ciliated cells of the efferent ducts, and in the peritubular stroma and blood vessels in the epididymis, and vas deferens. AQP9 was immunolocalized in the epithelial cells in the efferent ducts, epididymis and vas deferens. The morphology of Azara’s agouti testis excurrent ducts is similar to that reported for other rodents such as Cuniculus paca. The immunolocalization results of the AQPs suggest that the expression of AQPs is species-specific due to differences in localization and expression when compared to studies in other mammals species. The knowledge about the expression of AQPs in Azara’s agouti testis excurrent ducts is essential to support future reproductive studies on this animal, since previous studies show that AQPs may be biomarkers of male fertility and infertility.     

Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.     

Immunocytochemical localization of lipocalin-type prostaglandin D synthase in the bull testis and epididymis and on ejaculated sperm

Previously, we identified a 26-kDa fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase. The objective of the present study was to immunohistochemically localize this enzyme to the various cell types within the bull testis and seven subsegments of the epididymis, and on ejaculated sperm in order to gain further insight into its potential function in male reproduction. In the testis, immunoperoxidase staining was localized within the elongating spermatids and Sertoli cells of the seminiferous tubules, varying with the stage of the spermatogenic cycle. The highest level of staining occurred during stages III-VII. The cuboidal epithelial cells of the rete testis and efferent ducts were also immunoreactive. Expression of lipocalin-type prostaglandin D synthase was not uniform in the seven epididymal subsegments, suggesting a possible role in sperm maturation. In all epididymal regions, expression was limited to the epithelial principal cells; no immunoreactivity was apparent in other cell types. Lipocalin-type prostaglandin D synthase was strikingly localized in the caput epididymidis, while moderate to weak staining was observed in the remainder of the epididymis. Droplets of reaction product observed within the lumen increased progressively from the caput to cauda. Using fluorescence microscopy, we also localized lipocalin-type prostaglandin D synthase to the apical ridge of the acrosome on ejaculated sperm.     

Rabbit epididymal secretory proteins. I. Characterization and hormonal regulation

Analyses of samples of luminal fluid from the rete testis, distal efferent ducts, and epididymal regions 2-5 and 8 revealed that 91% of the fluid leaving the testis is reabsorbed by the efferent ducts, 79% of the remainder is reabsorbed proximal to epididymal regions 4 and 5, and there is a net secretion of fluid into the duct caudally. There is a net reabsorption by the efferent ducts of 73% of the protein leaving the testis and then a net secretion along the epididymis. SDS-PAGE of the luminal fluids indicated that four new protein bands that were not present in blood appeared in the efferent ducts, 5 in epididymal regions 1-5, 6 in regions 6 and 7, and one in region 8. Two bands in samples from the efferent ducts were absent caudally, and one band present in region 7 was absent in region 8. The rates of incorporation of (35)S-methionine into minced duct in vitro varied among regions when expressed per milligram of wet weight of tissue (region 2-5 > region 7 > region 6 > region 1 > region 8 > ductuli efferentes), and orchidectomy had little effect on the rates. Incorporation into four proteins that were secreted in vitro (M(r) 38 000, 20 000, 15 000, and 13 000) was reduced or abolished by orchidectomy and restored by testosterone therapy. The secretion of three proteins (M(r) 52 000, 23 000, and 22 000) was reduced or abolished by orchidectomy and not restored by testosterone therapy. SDS-PAGE of detergent extracts of sperm indicated that five proteins were lost and nine were gained during epididymal transit. Seven of the proteins gained were about the same molecular weight as proteins secreted by the epididymis (M(r) 94 000, 52 000, 38 000, 36 000, 22 000, 20 000, and 13 000) and were analyzed using N-terminal amino acid microsequencing.     

Role of epithelial cells of the male excurrent duct system of the rat in the endocytosis or secretion of sulfated glycoprotein-2 (clusterin)

The localization of sulfated glycoprotein-2 (clusterin; SGP-2) was investigated in the rete testis, efferent ducts, and epididymis of the rat using light (LM) and electron (EM) microscope immunocytochemistry. At the LM level, the epithelial cells of the rete testis and efferent ducts demonstrated an intense immunoperoxidase reaction over their apical and supranuclear regions, and sperm in the lumen of the efferent ducts were unreactive. In the EM, gold particles were found exclusively over the endocytic apparatus of these cells. In the proximal area of the epididymal initial segment, an insignificant immunostaining of epithelial cells and sperm was observed. However, the distal area of the initial segment showed a moderate staining over the epithelial principal cells and sperm, while in the intermediate zone of the epididymis a stronger reaction was observed over these cells. The strongest immunoperoxidase reaction was noted in the caput epididymidis, where it formed a distinct mottled pattern. Thus, while some principal cells were intensely stained, others were moderately or weakly stained; a few were completely unreactive. In the corpus and cauda epididymidis, the staining pattern was similar but not as intense. In the EM, only the secretory apparatus of these cells was found to be immunolabeled with gold particles. Sperm in the lumen of these different regions were also labeled. The epithelial clear cells were unreactive throughout the epididymis. Northern blot analysis substantiated these results and showed the presence of highest levels of SGP-2 mRNA in the caput epididymidis, especially in its proximal area, whereas increasingly lower levels were found in the corpus and cauda epididymidis. In summary, these results suggest that testicular SGP-2 dissociates from the sperm during passage through the rete testis and efferent ducts, where it is endocytosed by the epithelial cells lining these regions. In the epididymis, it is replaced by an epididymal SGP-2 that is secreted by the epithelial principal cells of the epididymis. Furthermore, in the epididymis, the principal cells appear to be in different functional states with respect to the secretion of epididymal SGP-2 within a given region of the duct as well as along the epididymal duct.     

Organization of tubules in the human caput epididymidis and the ultrastructure of their epithelia

The structure of the human caput epididymidis was examined by gross morphological and light and electron microscopic techniques. There were at least seven types of tubules, each characterized by a different epithelium. These tubules were connected with one another by at least eight types of junctions to form a network. Most of the caput epididymidis was composed of efferent ducts. Within these, five types of tubules, each with a different ciliated epithelium, were found in different regions; and four types of junctions between the efferent ducts and the epididymal tubule were observed. The efferent ducts left the testis, initially as parallel straight tubules containing both ciliated and non-ciliated cells in an epithelium of irregular height. Each efferent duct then coiled tortuously into lobules that folded over one another. These efferent ducts then branched out as thin tubules to join a network of dark tubules which were lined by a regular epithelium containing prominently vacuolated, non-ciliated cells. These tubules anastomosed via common cavities characterized by a ciliated cuboidal epithelium and sometimes joined tubules exhibiting a non-vacuolated ciliated epithelium. The latter, as well as typical efferent ducts, made connection with the epididymis proper in both end-to-end and end-to-side junctions. In the more distal junctions with the epididymis, the efferent ducts joined to a transitional epididymal ductule before joining to the side of the epididymis proper. Post-junctional epithelia in the beginning of the epididymis occasionally contained patches of cells characteristic of efferent ducts. Tall cells with long stereocilia constituted a discontinuous "initial segment"-like region of the epididymis. This is the most detailed study so far of the epithelia and the tubule organization in the caput epididymidis of any species, and most of the results are reported for the first time for the human. Although the pattern of the tubule network resembles that of some domestic species, the rich variety of epithelia has not been appreciated before.     

Segmental expression of the bradykinin type 2 receptor in rat efferent ducts and epididymis and its role in the regulation of aquaporin 9

Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.     

Mouse defensin beta 20 (Defb20) is expressed specifically in the caput region of the epididymis and regulated by androgen and testicular factors

Sperm cells undergo maturation during their transit throughout the epididymis. This process takes place in region-specific manner in which sperm are battered by proteins secreted by epithelium lining the epididymal duct. Most of the genes that encode for the proteins involved in the sperm maturation remain uncharacterized. Previous studies showed that family of β-defensins preferentially eaxpressed in male reproductive tracts and play an important role in both innate immunity and sperm fertility. In this study we characterized Defb20 to gain insight on its role in sperm maturation. Bioinformatic tools were used to analyzed functional domains and signal peptide. qRT-PCR analyses were used to analyzed tissue distribution, dependency on androgen and testicular factors and developmental-regulated expression analysis. Defb20 sequence contains important domains such as N-myristoilation and kinase binding sites which are putatively involved in the protein activation and protein-plasma membrane interaction. Moreover, DEFB20 contains a signal peptide indicating characteristic of secretory proteins. Defb20 was expressed exclusively in the epididymis with the highest expression in the caput region and was down-regulated by gonadectomy. Defb20 was also regulated by testicular factors in which the expression was down-regulated after efferent duct ligation (EDL). The dependency on the androgen was further confirmed by postnatal expression analysis in which Defb20 began to express at day-20 postnatal indicating specific stage of expression after initial development of the testis. In conclusion, Defb20 have a potential to be involved in the epididymal sperm maturation process.     

Two-dimensional electrophoresis of proteins in principal cells, spermatozoa, and fluid associated with the rat epididymis

Spermatozoa, fluids, and principal cells from different regions of the epididymis were characterized by two-dimensional electrophoresis. Rete testis fluid was collected after 36-h efferent duct ligation, and cauda epididymal fluid was collected by retrograde perfusion through the vas deferens. Spermatozoa were collected after their exudation from minced caput and corpus epididymal tissue. Principal cells were recovered after enzymatic disaggregation and centrifugal elutriation of epididymides. Two-dimensional polyacrylamide gel electrophoresis was used to prepare protein profiles of all samples. Comparison of the proteins found in rete testis fluid versus those found in cauda epididymal fluid revealed a dramatic change in composition, including the loss, addition, or retention of specific proteins as well as changes in the relative concentrations of certain proteins. Prominent cauda epididymal fluid proteins, possibly contributed by the epididymal epithelium, were detected at 16, 23, and 34 kDa. After epididymal transit, a considerable decrease was observed in the number of aqueous-soluble sperm proteins. Differences in the protein composition of epididymal epithelial principal cells from the caput versus corpus epididymidis were also noted, suggesting that functional differences exist for these epididymal regions. Of particular interest was the occurrence of a prominent protein of approximately 20-23 kDa found in all sperm samples, in fluids, and in caput and corpus principal cells. However, this protein was absent in cauda epididymal sperm after 36-h efferent duct ligation. The rapid loss of this protein from sperm after efferent duct ligation suggests that this surgical intervention may affect spermatozoa residing within the epididymis.     

Estrogen, efferent ductules, and the epididymis

Estrogen's presence in the male reproductive system has been known for over 60 years, but its potential function in the epididymis remains an important area of investigation. Estrogen is synthesized by germ cells, producing a relatively high concentration in rete testis fluid. There are two estrogen receptors (ESR), the presence of which in the head of the epididymis is well documented and consistent between species; however, in other regions of the epididymis, their expression appears to be isotype, species, and cell specific. ESR1 is expressed constitutively in the epididymis; however, its presence is downregulated by high doses of estrogen, making the design of experiments complicated, as the phenotype of the Cyp19a1(-/-) mouse does not resemble that of the Esr1(-/-) mouse. Ligand-independent and DNA-binding Esr1 mutant models further demonstrate the complexity and importance of both signaling pathways in maintenance of efferent ductules and epididymis. Data now reveal the presence of not only classical nuclear receptors, but also cytoplasmic ESR and rapid responding membrane receptors; however, their importance in the epididymis remains undetermined. ESR1 regulates ion transport and water reabsorption in the efferent ducts and epididymis, and its regulation of other associated genes is continually being uncovered. In the male, some genes, such as Aqp9 and Slc9a3, contain both androgen and estrogen response elements and are dually regulated by these hormones. While estrogen pathways are a necessity for fertility in the male, future studies are needed to understand the interplay between androgens and estrogens in epididymal tissues, particularly in cell types that contain both receptors and their cofactors.     

Expression and regulation of LRP‐2/megalin in epithelial cells lining the efferent ducts and epididymis during postnatal development

Low density lipoprotein receptor‐related protein‐2/megalin (LRP‐2) is a receptor belonging to the low density lipoprotein receptor family that mediates endocytosis and lysosomal degradation of a variety of ligands including apolipoprotein J (Apo J)/clusterin/SGP‐2. LRP‐2 has been shown to be expressed regionally in the adult rat epididymis. In this study, we describe the pattern of expression of LRP‐2 in the efferent ducts and epididymis during postnatal development of the rat and examine the role of testicular luminally derived substances on its expression. The expression of LRP‐2 was analyzed immunocytochemically in tissues of normal animals ranging in age from postnatal day 7–90 and in 15‐day‐old efferent‐duct‐ligated animals sacrificed at later ages. In the efferent ducts, LRP‐2 expression, appearing as a dense band on the apical surface of the nonciliated epithelial cells, was noted as early as day 7, well before the entry of sperm, Sertoli‐cell‐derived secretory products, and high levels of androgens. Efferent duct ligation studies further revealed that expression under this condition was comparable to controls at all later ages examined, suggesting that the factor regulating its expression was not a luminally derived testicular substance. In normal untreated animals, LRP‐2 expression was not apparent at any of the ages examined in the proximal initial segment of the epididymis. By comparison, the distal initial segment, although having no LRP‐2 expression from 7–15 days, showed expression in principal cells by day 21 which intensified at days 29 and 39. However, by day 49 and at later ages (56 and 90), LRP‐2 immunoreactivity over principal cells became spotty or with weak or moderate reactivity in some cells and none in others. LRP‐2 expression in the intermediate zone, proximal caput, corpus, and cauda regions also appeared in principal cells by day 21, intensified at days 29 and 39 and persisted as such at all later ages examined, correlating with high levels of androgens shown to occur by day 39. Although LRP‐2 expression in the distal caput region was evident in principal cells at days 21 and 29, it became spotty with weak, moderate, or absent reactivity over principal cells at all later ages. These data suggest that LRP‐2 expression is under the influence of both stimulatory and region‐specific inhibitory factors. Analysis of 15‐day‐old efferent‐duct‐ligated animals at all later ages examined revealed that there was no change in LRP‐2 expression along the entire epididymis, suggesting that both the stimulatory and inhibitory factors are not luminally derived testicular substances. The observed pattern of LRP‐2 expression in all regions of the epididymis, except the distal caput region, was similar to that described for Apo J internalization by principal cells during postnatal development, showing a correlation between LRP‐2 expression and its ligand, Apo J. In summary, LRP‐2 expression in the epididymis undergoes region‐specific changes during postnatal development and appears to be influenced by both stimulatory and inhibitory factors. Mol. Reprod. Dev. 53:282–293, 1999. © 1999 Wiley‐Liss, Inc.