Crucial Roles of the Mesenchymal Androgen Receptor in Wolffian Duct Development.

Wolffian duct (WD) maintenance and differentiation is predominantly driven by the androgen action, which is mediated by the androgen receptor (AR). It is well established that the mesenchyme indicates the fate and differentiation of epithelial cells. However, in vivo developmental requirement of mesenchymal AR in WD development is still undefined. By designing a mesenchyme-specific Ar knockout (ARcKO), we discovered that the loss of mesenchymal Ar led to the bilateral or unilateral degeneration of caudal WDs and cystic formation at the cranial WDs. Ex vivo culture of ARcKO WDs invariably resulted in bilateral defects, suggesting that some factor(s) originating from surrounding tissues in vivo might promote WD survival and growth even in the absence of mesenchymal Ar. Mechanistically, we found cell proliferation was significantly reduced in both epithelial and mesenchymal compartments; but cell apoptosis was not affected. Transcriptomic analysis by RNA sequencing of E14.5 mesonephroi revealed 131 differentially expressed genes. Multiple downregulated genes (Top2a, Wnt9b, Lama2, and Lamc2) were associated with morphological and cellular changes in ARcKO male embryos (ie, reduced cell proliferation and decreased number of epithelial cells). Mesenchymal differentiation into smooth muscle cells that are critical for morphogenesis was also impaired in ARcKO male embryos. Taken together, our results demonstrate the crucial roles of the mesenchymal AR in WD maintenance and morphogenesis in mice.

Ift25 is not a cystic kidney disease gene but is required for early steps of kidney development.

Eukaryotic cilia are assembled by intraflagellar transport (IFT) where large protein complexes called IFT particles move ciliary components from the cell body to the cilium. Defects in most IFT particle proteins disrupt ciliary assembly and cause mid gestational lethality in the mouse. IFT25 and IFT27 are unusual components of IFT-B in that they are not required for ciliary assembly and mutant mice survive to term. The mutants die shortly after birth with numerous organ defects including duplex kidneys. Completely duplex kidneys result from defects in ureteric bud formation at the earliest steps of metanephric kidney development. Ureteric bud initiation is a highly regulated process involving reciprocal signaling between the ureteric epithelium and the overlying metanephric mesenchyme with regulation by the peri-Wolffian duct stroma. The finding of duplex kidney in Ift25 and Ift27 mutants suggests functions for these genes in regulation of ureteric bud initiation. Typically the deletion of IFT genes in the kidney causes rapid cyst growth in the early postnatal period. In contrast, the loss of Ift25 results in smaller kidneys, which show only mild tubule dilations that become apparent in adulthood. The smaller kidneys appear to result from reduced branching in the developing metanephric kidney. This work indicates that IFT25 and IFT27 are important players in the early development of the kidney and suggest that duplex kidney is part of the ciliopathy spectrum.

Novel function of LHFPL2 in female and male distal reproductive tract development.

Congenital reproductive tract anomalies could impair fertility. Female and male reproductive tracts are developed from Müllerian ducts and Wolffian ducts, respectively, involving initiation, elongation and differentiation. Genetic basis solely for distal reproductive tract development is largely unknown. Lhfpl2 (lipoma HMGIC fusion partner-like 2) encodes a tetra-transmembrane protein with unknown functions. It is expressed in follicle cells of ovary and epithelial cells of reproductive tracts. A spontaneous point mutation of Lhfpl2 (LHFPL2(G102E)) leads to infertility in 100% female mice, which have normal ovarian development, ovulation, uterine development, and uterine response to exogenous estrogen stimulation, but abnormal upper longitudinal vaginal septum and lower vaginal agenesis. Infertility is also observed in ~70% mutant males, which have normal mating behavior and sperm counts, but abnormal distal vas deferens convolution resulting in complete and incomplete blockage of reproductive tract in infertile and fertile males, respectively. On embryonic day 15.5, mutant Müllerian ducts and Wolffian ducts have elongated but their duct tips are enlarged and fail to merge with the urogenital sinus. These findings provide a novel function of LHFPL2 and a novel genetic basis for distal reproductive tract development; they also emphasize the importance of an additional merging phase for proper reproductive tract development.

Fibroblast growth factor receptor signaling in kidney and lower urinary tract development.

Fibroblast growth factor receptors (FGFRs) and FGF ligands are highly expressed in the developing kidney and lower urinary tract. Several classic studies showed many effects of exogenous FGF ligands on embryonic renal tissues in vitro and in vivo. Another older landmark publication showed that mice with a dominant negative Fgfr fragment had severe renal dysplasia. Together, these studies revealed the importance of FGFR signaling in kidney and lower urinary tract development. With the advent of modern gene targeting techniques, including conditional knockout approaches, several publications have revealed critical roles for FGFR signaling in many lineages of the kidney and lower urinary tract at different stages of development. FGFR signaling has been shown to be critical for early metanephric mesenchymal patterning, Wolffian duct patterning including induction of the ureteric bud, ureteric bud branching morphogenesis, nephron progenitor survival and nephrogenesis, and bladder mesenchyme patterning. FGFRs pattern these tissues by interacting with many other growth factor signaling pathways. Moreover, the many genetic Fgfr and Fgf animal models have structural defects mimicking numerous congenital anomalies of the kidney and urinary tract seen in humans. Finally, many studies have shown how FGFR signaling is critical for kidney and lower urinary tract patterning in humans.

Non-canonical Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension.

Congenital anomalies of the kidney and urinary tract (CAKUT) affect about 1 in 500 births and are a major cause of morbidity in infants. Duplex collecting systems rank among the most common abnormalities of CAKUT, but the molecular basis for this defect is poorly understood. In mice, conditional deletion of Wnt5a in mesoderm results in bilateral duplex kidney and ureter formation. The ureteric buds (UBs) in mutants emerge as doublets from the intermediate mesoderm (IM)-derived nephric duct (ND) without anterior expansion of the glial cell line-derived neurotrophic factor (Gdnf) expression domain in the surrounding mesenchyme. Wnt5a is normally expressed in a graded manner at the posterior end of the IM, but its expression is down-regulated prior to UB outgrowth at E10.5. Furthermore, ablation of Wnt5a in the mesoderm with an inducible Cre at E7.5 results in duplex UBs, whereas ablation at E8.5 yields normal UB outgrowth, demonstrating that Wnt5a functions in IM development well before the formation of the metanephros. In mutants, the posterior ND is duplicated and surrounding Pax2-positive mesenchymal cells persist in the nephric cord, suggesting that disruption of normal ND patterning prompts the formation of duplex ureters and kidneys. Ror2 homozygous mutants, which infrequently yield duplex collecting systems, show a dramatic increase in incidence with the additional deletion of one copy of Wnt5a, implicating this receptor in non-canonical Wnt5a signaling during IM development. This work provides the first evidence of a role of Wnt5a/Ror2 signaling in IM extension and offers new insights into the etiology of CAKUT and possible involvement of Wnt5a/Ror2 mutations.

Stage specific requirement of Gfrα1 in the ureteric epithelium during kidney development.

Glial cell line-derived neurotrophic factor (GDNF) binds a coreceptor GDNF family receptor α1 (GFRα1) and forms a signaling complex with the receptor tyrosine kinase RET. GDNF-GFRα1-RET signaling activates cellular pathways that are required for normal induction of the ureteric bud (UB) from the Wolffian duct (WD). Failure of UB formation results in bilateral renal agenesis and perinatal lethality. Gfrα1 is expressed in both the epithelial and mesenchymal compartments of the developing kidney while Ret expression is specific to the epithelium. The biological importance of Gfrα1's wider tissue expression and its role in later kidney development are unclear. We discovered that conditional loss of Gfrα1 in the WD epithelium prior to UB branching is sufficient to cause renal agenesis. This finding indicates that Gfrα1 expressed in the nonepithelial structures cannot compensate for this loss. To determine Gfrα1's role in branching morphogenesis after UB induction we used an inducible Gfrα1-specific Cre-deletor strain and deleted Gfrα1 from the majority of UB tip cells post UB induction in vivo and in explant kidney cultures. We report that Gfrα1 excision from the epithelia compartment after UB induction caused a modest reduction in branching morphogenesis. The loss of Gfrα1 from UB-tip cells resulted in reduced cell proliferation and decreased activated ERK (pERK). Further, cells without Gfrα1 expression are able to populate the branching UB tips. These findings delineate previously unclear biological roles of Gfrα1 in the urinary tract and demonstrate its cell-type and stage-specific requirements in kidney development.

FGF8 is essential for formation of the ductal system in the male reproductive tract.

During development of the urogenital tract, fibroblast growth factor 8 (Fgf8) is expressed in mesonephric tubules, but its role in this tissue remains undefined. An evaluation of previously generated T-Cre-mediated Fgf8-deficient mice (T-Cre; Fgf8(flox/Δ2,3) mice), which lack Fgf8 expression in the mesoderm, revealed that the cranial region of the Wolffian duct degenerated prematurely and the cranial mesonephric tubules were missing. As a result, the epididymis, vas deferens and efferent ductules were largely absent in mutant mice. Rarb2-Cre was used to eliminate FGF8 from the mesonephric tubules but to allow expression in the adjacent somites. These mutants retained the cranial end of the Wolffian duct and formed the epididymis and vas deferens, but failed to elaborate the efferent ductules, indicating that Fgf8 expression by the mesonephric tubules is required specifically for the formation of the ductules. Ret knockout mice do not form the ureteric bud, a caudal outgrowth of the Wolffian duct and progenitor for the collecting duct network in the kidney, but they do develop the cranial end normally. This indicates that Fgf8, but not Ret, expression is essential to the outgrowth of the cranial mesonephric tubules from the Wolffian duct and to the development of major portions of the sex accessory tissues in the male reproductive tract. Mechanistically, FGF8 functions upstream of Lhx1 expression in forming the nephron, and analysis of Fgf8 mutants similarly shows deficient Lhx1 expression in the mesonephric tubules. These results demonstrate a multifocal requirement for FGF8 in establishing the male reproductive tract ducts and implicate Lhx1 signaling in tubule elongation.

The critical time window for androgen-dependent development of the Wolffian duct in the rat.

Androgens are thought to separately regulate stabilization and differentiation of the Wolffian duct (WD), but the time windows for these effects are unclear. To address this, fetal rats were exposed to flutamide within either an early window (EW) [embryonic day 15.5 (E15.5) to E17.5], when the WD degenerates in the female, or a later window (LW) (E19.5-E21.5), when the WD morphologically differentiates in the male, or during the full window of WD development (FW) (E15.5-21.5). WDs were examined for abnormalities during fetal (E21.5) or postnatal life, and anogenital distance and prostate presence/absence were recorded. Exposure to FW- or EW-flutamide, but not to LW-flutamide, induced comparable abnormalities in the fetal WD at E21.5, namely reduced WD coiling, reduced cell proliferation, reduced epithelial cell height, altered epithelial vimentin expression, and reduced expression of smooth muscle actin in the WD inner stroma. Exposure to EW- or FW-flutamide, but not to LW-flutamide, resulted in incomplete/absent WDs in more than 50% of males by adulthood, although such abnormalities were infrequent in fetal life. These findings suggest that androgen action during the EW is sufficient to promote WD morphological differentiation several days later. Because the androgen receptor is expressed in the WD stroma but not in the epithelium during this EW, WD differentiation is likely to be dependent on androgen-mediated signaling from the stroma to the epithelium. In conclusion, the critical window for androgen action in regulating WD development in the rat is between E15.5 and E17.5. This window is also important for prostate formation and anogenital distance masculinization.

Androgen-dependent mechanisms of Wolffian duct development and their perturbation by flutamide.

Androgens play a vital role in Wolffian duct (WD) development, but the mechanisms that underlie this are unknown. The present study used in utero exposure of pregnant rats to the androgen receptor antagonist flutamide (50 or 100 mg/kg) to explore possible mechanisms. Pregnant rats were treated from embryonic d 15.5 (E15.5), and WDs were isolated from fetuses from E17.5-E21.5 and from adults. WD morphology was evaluated, and total length of the duct lumen was determined in fetal samples. Fetal WDs were immunostained for androgen receptor and stromal (inner and outer) and/or epithelial-cell-specific markers and analyzed for cell proliferation and apoptosis. In adulthood, most flutamide-exposed males lacked proximal WD-derived tissues, whereas at E18.5-E19.5, a time when the WD has completely regressed in females, a complete normal WD was present in all flutamide-exposed animals. This suggests that flutamide, at doses of 50 or 100 mg/kg, interferes with WD differentiation, not stabilization. Consistent with this, WD elongation/coiling increased in controls by 204% between E19.5 and E21.5 but increased less significantly (103%) in flutamide-exposed animals. This was associated with reduced cell proliferation, but there was no increase in apoptosis or change in expression of androgen receptor mRNA or protein. Flutamide treatment impaired differentiation of inner stromal cells, shown by decreased expression of smooth muscle actin, before effects were noted in the epithelium, consistent with androgens driving WD development via stromal-epithelial interactions. In conclusion, WD differentiation is far more susceptible to blockade of androgen action than is its initial stabilization, and these effects may be mediated by disruption of stromal-epithelial interactions.

MSX2 promotes vaginal epithelial differentiation and wolffian duct regression and dampens the vaginal response to diethylstilbestrol.

In utero exposure to diethylstilbestrol (DES) leads to patterning defects in the female reproductive tract (FRT) and a propensity to the development of vaginal adenocarcinomas in humans. In the mouse, DES treatment similarly induces a plethora of FRT developmental defects, including stratification of uterine epithelium and presence of glandular tissue in cervix and vagina. Uterine abnormalities are associated with repression of the homeobox gene Msx2, and DES leads to an altered uterine response in Msx2 mutants including a dilated uterine lumen. Here we investigate the role of Msx2 in normal vaginal development and in FRT response to DES. During vaginal development, Msx2 is required for Tgfbeta2 and Tgfbeta3 expression and for proper vaginal epithelial differentiation. Moreover, Msx2 is involved in caudal Wolffian duct regression by promoting apoptosis. Consistently, neonatal DES exposure represses Msx2 expression in the Wolffian duct epithelium and inhibits its apoptosis and subsequent regression. Intriguingly, although DES treatment also represses Msx2 expression in the vaginal epithelium, a much more severe DES-induced vaginal phenotype was observed in Msx2 mutant mice, including a complete failure of Müllerian vaginal epithelial stratification and a severely dilated vaginal lumen, accompanied by loss of p63 and water channel protein expression. These results demonstrate a critical role for Msx2 in counteracting the effect of DES on FRT patterning and suggest that the response to DES may be highly variable depending on the genotype of an individual.

Changes in gene expression during Wolffian duct development.

BACKGROUND: Wolffian ducts (WDs) are the embryonic precursors of the male reproductive tract. Their development is induced by testosterone, which interacts with the androgen receptor (AR). The molecular pathways underlying androgen-dependent WD development are largely unknown. We aimed to identify AR target genes important in this process. METHODS: RNA was isolated from rat WDs at E17.5 and E20.5. Affymetrix GeneChip expression arrays were used to identify transcripts up- or downregulated more than 2-fold. Regulation of seven transcripts was confirmed using quantitative PCR. RESULTS: Transcripts from 76 known genes were regulated, including modulators of insulin-like growth factor and transforming growth factor-beta signalling. By controlling these modulators, androgens may indirectly affect growth factor signalling pathways important in epithelial-mesenchymal interactions and organ development. Caveolin-1, also upregulated, may play a role in modifying as well as mediating AR signalling. Differentiation of WD epithelium and smooth muscle, innervation and extracellular matrix synthesis were reflected in regulation of other transcripts. Several genes were previously suggested to be regulated by androgens or contained functional or putative androgen/glucocorticoid response elements, indicating they may be direct targets of androgen signalling. CONCLUSION: Our results suggest novel cohorts of signals that may contribute to androgen-dependent WD development and provide hypotheses that can be tested by future studies.

Effects of steroids and dioxin (2,3,7,8-TCDD) on the developing wolffian ducts of the tiger salamander (Ambystoma tigrinum).

This study was undertaken to investigate effects of the prototypical dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on steroid-dependent development of the wolffian ducts of an amphibian, the tiger salamander (Ambystoma tigrinum). Larvae with immature gonads and undeveloped mullerian ducts were injected with the steroid hormones estradiol (E2), dihydrotestosterone (DHT), or vehicle alone. Additionally, steroid-treated and vehicle-control larvae were immersed in sub-lethal solutions of technical grade TCDD (0, 0.0003, 0.003, 0.03, 0.3, and 3.0 microg TCDD/L). Both steroid treatments stimulated hypertrophy of the wolffian duct epithelium and an increase in mean epithelial cell size. Only DHT treatment stimulated epithelial cell proliferation. TCDD stimulated wolffian duct hypertrophy through an increase in mean epithelial cell size. TCDD acted as an androgen agonist on wolffian duct epithelial area and epithelial cell size. TCDD had no effect on wolffian duct epithelium among E2-injected animals. Stimulatory effects on cell size were observed at 0.0003 microg/L TCDD in saline-injected animals and at 0.003 microg/L TCDD in DHT-injected animals. Both E2 and DHT stimulated growth of the wolffian ducts early in development. Technical grade TCDD alone mimics E2 and DHT action but exhibits an androgen-agonistic action in the presence of exogenously administered DHT. Implications of possible interactions between TCDD and xenosteroids are discussed.

Interactions of androgens and estradiol on sex accessory ducts of larval tiger salamanders, Ambystoma tigrinum.

Immature tiger salamander larvae were treated with 12.5 or 25 micrograms of estradiol, testosterone, or dihydrotestosterone (DHT), or 12.5 micrograms of estradiol combined with 12.5 micrograms of either testosterone or DHT. Müllerian duct epithelium was more stimulated by combined steroid treatment than by any steroid alone. Estradiol antagonized the action of DHT in the Wolffian duct. Both of the androgens and estradiol when administered alone at the higher dose stimulated enlargement of connective tissue surrounding the ducts, but the combined 12.5 micrograms androgen/12.5 micrograms estrogen treatment was more effective even though the total steroid administered was the same. The effectiveness of DHT on müllerian cells of this species is evidence against a required aromatization of androgens to explain paradoxical steroid effects and suggests that fundamental differences may exist in steroid receptors of müllerian ducts, connective tissue, and Wolffian ducts. A possible role for the urodele duct system for assessing estrogenic activity of environmental contaminants is discussed.

Posterior extension of the chick nephric (Wolffian) duct: the role of fibronectin and NCAM polysialic acid.

The nephric duct of the chick embryo starts to form at about stage 10 of Hamburger and Hamilton ([1951] J. Morphol. 88:49-92) and extends posteriorly, fusing with the cloaca at about the end of the third day of incubation (HH stage 17). Evidence from the literature suggests that the extension involves active migration of the posterior tip. This investigation concerned some molecules that might control this migration: fibronectin, vitronectin, the beta 1 integrin receptor, and NCAM polysialic acid. The concentration of fibronectin in the extracellular matrix was found by immunocytochemistry to be negligible at the posterior end of the duct; treatment of the living embryo with GRGDS failed to halt further extension of the duct; SEM examination of embryos treated with the synthetic peptides of fibronectin GRGDS, GRDGS, SDGR, and GRGES, or with vitronectin, revealed negligible morphological effects on the duct. It is concluded that there is yet no evidence that fibronectin is an important factor in duct migration. NCAM polysialic acid had a similar distribution to fibronectin, but treatment of the living embryo with Endo-N caused cessation of extension of the duct. Endo-N is an enzyme that specifically degrades PSA without affecting the NCAM polypeptide itself. It is suggested therefore that PSA may play an important role in duct extension. The synthetic peptides of fibronectin each produced distinctive patterns of blebbing on the surfaces of cells in trunk mesoderm, but the duct cells were unaffected. GRGES and SDGR caused blebbing on cells in the somites and the anterior segmental plate, though not on cells in the posterior segmental plate. This suggests that integrin receptors change in the anterior segmental plate as the mesoderm forms somites from somitomeres.

Anti-masculinizing action of estradiol and cyproterone acetate: regulation of a protein fraction with phospholipase-A2 stimulatory and masculinizing activities.

Recently we have identified a protein fraction (55-63 K) from male and testosterone-exposed female mouse genital tract, which stimulates phospholipase A2 (PLA2) and induces masculine differentiation in an undifferentiated mouse genital explant, suggesting a role of this protein in the action of testosterone. In the current study we have further investigated the role of this protein by determining whether anti-masculinizing agents, namely, estradiol and cyproterone acetate, have any effect on the production of this protein. The results described here indicate that a protein fraction containing PLA2 stimulatory activity was present in both control male and estradiol- or cyproterone acetate-exposed male fetal genital tract. However the specific activity of the PLA2-stimulatory protein was significantly higher in the control males than in the experimental males. We did not find any major difference in the behavior of this protein fraction in various chromatographic steps except that in CM-sepharose column; the PLA2-stimulatory activity from the male preparation was eluted in two overlapping peaks with 0.3 and 0.25 M NaCl and that from the treated males was eluted only with 0.25 M NaCl. The SDS-gel analysis of this protein fraction revealed a doublet band (55 and 63 K) in control samples and primarily a 63 K band in experimental samples. The protein fraction from all these sources showed a significant difference in their biological activity. The control male preparation induced Wolffian duct whereas the estradiol sample was completely ineffective and the cyproterone acetate sample was partially effective in inducing Wolffian duct. Thus, it appears that the protein fraction has a role in the masculinizing action of testosterone.

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. II. Instructive induction of Wolffian duct epithelia by neonatal seminal vesicle mesenchyme.

When grown as renal grafts in adult male hosts, the upper (cranial), middle and lower (caudal) portions of fetal mouse and rat Wolffian ducts developed into epididymis, epididymis plus ductus deferens, and seminal vesicle, respectively. In heterotypic tissue recombinants, the epithelia from upper and middle Wolffian ducts were instructively induced to undergo seminal vesicle morphogenesis by neonatal seminal vesicle mesenchyme. Functional cytodifferentiation was examined in these recombinants using antibodies against major androgen-dependent, seminal vesicle-specific secretory proteins. The instructively induced Wolffian duct epithelia synthesized normal amounts of all of the secretory proteins characteristic of mature seminal vesicles, as judged by immunocytochemistry on tissue sections and gel electrophoresis plus immunoblotting of secretions extracted from the recombinants. In heterospecific recombinants composed of rat and mouse tissues, the seminal vesicle proteins induced were specific for the species that had provided the epithelium. This showed that the seminal vesicle epithelium in the recombinants was derived from instructively induced Wolffian duct epithelium and not from epithelial contamination of the mesenchymal inductor. Upper Wolffian duct epithelium, instructively induced to undergo seminal vesicle morphogenesis, did not express epididymis-specific secretory proteins, showing that its normal development had been simultaneously repressed.

Influence of the immature testis on sexual differentiation in the tammar wallaby, Macropus eugenii (Macropodidae: Marsupialia).

Reproduction in the tammar wallaby, Macropus eugenii (Desmarest), is highly seasonal in the females but not the males. This study was designed to determine whether the difference is established during early life as a result of exposure to the developing testes. At day 10 after birth, when the sex can be distinguished externally, testes were removed from males and placed under the flank skin of females, while other groups of males and females were subjected to surgery without interfering with the gonads. The testis grafts remained palpable for 3-6 months. Sex-chromosome constitution was confirmed by karyotyping. At 3 years of age, the body weights and dimensions of the grafted females were not significantly different from those of the sham-operated females, whereas those of the castrated males were significantly larger and were equal to those of the sham-operated males, indicating that there is genetical control of growth independent of the testis in this species of marsupial. During 5 years of observations, none of the grafted females ever produced young, whereas all of the sham-operated females produced young each year from the second year. The grafted females had a mixture of male and female reproductive structures. The pouch and mammary glands developed normally, as did the Mullerian duct derivatives, the vaginal complex, the uteri and the oviducts. The ovaries were either devoid of oocytes and follicles or had reduced numbers, the Wolffian ducts were retained to varying degrees, the urogenital strand had developed into a prostate indistinguishable in size and structure from that of intact males, and the genital tubercle had developed into a normal-sized penis with a crus penis and Cowper's glands. In the castrated males, the scrotum developed normally and contained the gubernaculum and vas deferens. There was no evidence of Mullerian duct derivatives, and the urogenital strand was a simple canal, as in females. There were no Cowper's glands and no penis or erectile tissue. In one hemicastrated male, there was no development of the penis, although the remaining testis occupied the scrotum and showed compensatory hypertrophy. These findings indicate that the testis, at day 10, has a profound influence on the early differentiation of the Wolffian ducts, prostate and penis but cannot influence the differentiation of the Mullerian duct derivatives. The testis does not have any effect on the development of the pouch, mammary glands or scrotum or on somatic growth, all of which are apparently under independent genetical control.