The bone morphogenetic proteins are members of the transforming growth factor-beta superfamily . Ozkaynak et al. (1990) purified a novel bovine osteogenic protein homolog, which they termed 'osteogenic
protein-1' (OP1).
NCBI Summary:
This gene encodes a secreted ligand of the TGF-beta (transforming growth factor-beta) superfamily of proteins. Ligands of this family bind various TGF-beta receptors leading to recruitment and activation of SMAD family transcription factors that regulate gene expression. The encoded preproprotein is proteolytically processed to generate each subunit of the disulfide-linked homodimer, which plays a role in bone, kidney and brown adipose tissue development. Additionally, this protein induces ectopic bone formation and may promote fracture healing in human patients. [provided by RefSeq, Jul 2016]
General function
Ligand, Growth factor
Comment
Bone morphogenetic proteins (BMP) -4, -6 and -7 potently suppress basal and LH-induced androgen production by bovine theca interna cells in primary culture: could ovarian hyperandrogenic dysfunction be caused by a defect in thecal BMP signaling?
Glister C, et al 2005 .
We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and -7) while bovine granulosa cells and oocytes express BMP-6. Therefore BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, -6 and -7 potently suppress both basal (P < 0.0001; respective IC50 values 0.61, 0.25, 1.13ng/ml) and LH-induced (P < 0.0001; respective IC50 values 5.00. 0.55, 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semi-quantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17 levels of mRNA encoding StAR and P450scc and 3betaHSD were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein following BMP treatment (P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by co-incubating cells with BMPs and several potential BMP antagonists, chordin, gremlin and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and -7 respectively and the observation that both antagonists enhanced (P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP-4 and -7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be due to a defective autoregulatory pathway involving thecal BMP signaling.
Interaction of ovarian steroidogenesis and clock gene expression modulated by bone morphogenetic protein-7 in human granulosa cells. Nagao S et al. (2018) A functional link between clock gene expression and ovarian steroidogenesis was studied using human granulosa KGN cells. Similarities between changes in the mRNA and protein expression levels of Bmal1 and Clock and those of Per2 and Cry1 were found in KGN cells after treatment with forskolin. Among the interrelationships between the expression levels of clock and steroidogenic factors, Clock mRNA had a strongly positive correlation with P450arom and a negative correlation with 3βHSD. Knockdown of Clock gene by siRNA resulted in a significant reduction of estradiol production by inhibiting P450arom expression, while it induced a significant increase of progesterone production by upregulating 3βHSD in KGN cells treated with forskolin. Moreover, BMP-7 had an enhancing effect on the expression of Clock mRNA and protein in KGN cells. Thus, the expression levels of Clock, being upregulated by forskolin and BMP-7, were functionally linked to estradiol production and progesterone suppression by human granulosa cells.//////////////////BMP4 and BMP7 suppress StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 in human granulosa-lutein cells. Zhang H et al. (2015) Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pre-treatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1), but not ALK4/5/7 (SB-431542). Moreover, siRNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD1/5/8 was reversed by treatment with DMH1 or siRNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.//////////////////
Bone Morphogenetic Protein 7 Increased Vascular Endothelial Growth Factor (VEGF)-A Expression in Human Granulosa Cells and VEGF Receptor Expression in Endothelial Cells. Akiyama I 2013 et al.
The formation of an individual capillary network in the theca cell layer is required for ovarian folliculogenesis. Although vascular endothelial growth factor (VEGF) is critical for this process, the regulation of VEGF has been unclear. In the present study, the relationship between VEGF and intraovarian cytokine, bone morphogenetic protein 7 (BMP-7) was investigated. Granulosa cells (GC), obtained from in vitro fertilization patients, were cultured with BMP-7 followed by RNA extraction. Human umbilical vein endothelial cells (HUVECs) were also cultured with BMP-7 followed by RNA extraction, tube formation assay, or cell count analysis. The BMP-7 stimulated VEGF messenger RNA (mRNA) and protein expression in GC significantly. In HUVEC, BMP-7 increased an approximately 1.8-fold in the cell number and induced the tube formation significantly compared to control. The BMP-7 also induced a 2-fold increase in VEGF receptor mRNA transcript relative abundance in HUVEC. The BMP-7, a theca cell-derived factor, may stimulate endothelial cell to form vasculature in the follicle via 2 distinct mechanisms, induction of VEGF expression in GC and increased sensitivity of endothelial cells to VEGF.
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Bone morphogenetic protein 4 (BMP-4) and BMP-7 induce vascular endothelial growth factor expression in bovine granulosa cells. Shimizu T et al. Bone morphogenetic protein-4 (BMP-4) and BMP-7, theca cell-derived growth factors, directly affect the granulosa cell function. The aim of this study was to examine the involvement of BMP-4 or BMP-7 in vascular endothelial growth factor (VEGF) expression in bovine granulosa cells. Granulosa cells were collected from small follicles (4-6 mm) and seeded at a density of 2-5 ?10(5) cells per well in Dulbecco's modified Eagle's medium (DMEM)/F12 medium with BMP-4 or BMP-7. The expression of VEGF messenger RNA and protein was the maximum when 1.0 ng/mL of BMP-4 was added to the culture medium. On the other hand, 10 ng/mL of BMP-7 significantly increased the expression of the VEGF gene and protein. In addition, BMP-4 stimulated the expression of Smad1 and Smad5 genes in granulosa cells, whereas BMP-7 stimulated the expression of Smad5 gene. These results suggested that BMP-4 and BMP-7 may be associated with VEGF expression via several specific Smads in bovine granulosa cells: BMP-4 via Smad1/Smad5 and BMP-7 via Smad5. In conclusion, theca cell-derived BMP-4 and BMP-7 might contribute to follicular vasculature and development by inducing VEGF expression in granulosa cells.
Woo-Sik Lee et al 2001 Effect of Bone Morphogenetic Protein-7 on
Folliculogenesis and Ovulation in the Rat.
BMP-7 enhanced P450 aromatase (P450arom) but suppressed steroidogenic acute
regulatory protein (StAR) mRNAs induced by FSH, whereas mRNAs encoding further-downstream steroidogenic
enzymes, including P450 side-chain cleavage enzyme and 3?hydroxysteroid dehydrogenase, were not significantly
altered. These findings suggest that BMP-7 stimulation and inhibition of P450arom and StAR mRNA expression,
respectively, may play a role in the mechanisms underlying the differential regulation of estradiol and progesterone
production. Ovaries treated with BMP-7 had decreased numbers of
primordial follicles, yet had increased numbers of primary, preantral, and antral follicles, suggesting that BMP-7 may
act to facilitate the transition of follicles from the primordial stage to the pool of primary, preantral, and antral follicles.
BMP-7 caused an increase in DNA synthesis and proliferation of granulosa cells
from small antral follicles in vitro. In contrast to the stimulatory activity, BMP-7 exhibited pronounced inhibitory
effects on ovulation rate and serum progesterone levels. These findings establish important new biological activities of
BMP-7 in the context of ovarian physiology, including folliculogenesis and ovulation.
Effects of bone morphogenetic protein-7 (BMP-7) on primordial follicular growth in the mouse ovary.
Lee WS, et al 2004 .
Previously, bone morphogenetic protein-7 (BMP-7) was suggested as a factor that may act to facilitate the transition of follicles from primordial stage to the pool of developed primary, preantral, and antral follicles (Lee et al. 2001: Biol Reprod 65:994-999.). Thus, aim of the present study was to evaluate effect(s) of BMP-7 on the primordial-primary follicle transition. Neonatal mouse ovaries were cultured in the presence or absence of 100 mIU/ml FSH with various doses of BMP-7 (0, 10, and 100 ng/ml). After 4-day culture period, number of follicles was counted and the expression of transcripts for FSH receptor (FSHR), kit ligand (KL), and c-kit was measured by RT-PCR. BMP-7 alone at 100 ng/ml concentration stimulated follicle development with concurrent increase of mRNA for FSHR. BMP-7 alone down-regulated KL expression however, the ratio between KL1 and KL2 was increased. There was no change in the c-kit mRNA expression. Results of the present study suggest that the BMP-7 is one of the factors involved in primordial-primary follicle transition in the mouse ovary and it may play a role in expression of FSHR for further follicular development. Mol. Reprod. Dev. 69: 159-163, 2004. Bone Morphogenetic Proteins (BMP) -4, -6, and -7 Potently Suppress Basal and Luteinizing Hormone-Induced Androgen Production by Bovine Theca Interna Cells in Primary Culture: Could Ovarian Hyperandrogenic Dysfunction Be Caused by a Defect in Thecal BMP Signaling?
Glister C, et al
We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and -7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, -6, and -7 potently suppress both basal (P < 0.0001; respective IC(50) values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced (P < 0.0001; respective IC(50) values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3beta-hydroxy- steroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment (P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and -7, respectively, and the observation that both antagonists enhanced (P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP-4 and -7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.Differential regulation of steroidogenesis by bone morphogenetic proteins in granulosa cells: Involvement of ERK signaling and oocyte actions in FSH-induced estrogen production. Miyoshi T et al. In the present study we investigated the cellular mechanism by which oocytes and BMPs govern FSH-induced steroidogenesis using rat primary granulosa cells. BMP-6 and BMP-7 both inhibited FSH- and forskolin-induced progesterone synthesis and reduced cAMP synthesis independent of the presence or absence of oocytes. BMP-7 also increased FSH-induced estradiol production, and the response was further augmented in the presence of oocytes. In contrast, BMP-6 had no impact on estradiol synthesis regardless of the presence of oocytes. Since BMP-7 changed neither forskolin- nor cAMP-induced estradiol production, the BMP-7 action was mediated through a FSH receptor signaling mechanism that was independent of cAMP-PKA pathway. Treatment with FSH, but not cAMP, activated ERK1/2 phosphorylation in granulosa cells, which was further accelerated by oocytes. A specific ERK inhibitor U0126 increased estradiol production and decreased FSH- and forskolin-induced progesterone production and cAMP synthesis. This suggests that ERK activation is directly linked to inhibition of estradiol synthesis and amplification of cAMP. Moreover, FSH-induced ERK1/2 phosphorylation was inhibited by BMP-7 but not influenced by BMP-6. On the other hand, BMP signaling including Smad1/5/8 phosphorylation and Id-1 transcription was upregulated by FSH and oocytes in granulosa cells through inhibition of Smad6/7 expression. Collectively, oocytes enhance FSH-induced MAPK activation and BMP signaling in granulosa cells, which leads to differential regulation of steroidogenesis elicited by BMPs in the presence of FSH in developing follicles.
Differential regulation of steroidogenesis by bone morphogenetic proteins in granulosa cells: Involvement of ERK signaling and oocyte actions in FSH-induced estrogen production.Miyoshi T, Otsuka F, Inagaki K, Otani H, Takeda M, .
In the present study we investigated the cellular mechanism by which oocytes and BMPs govern FSH-induced steroidogenesis using rat primary granulosa cells. BMP-6 and BMP-7 both inhibited FSH- and forskolin-induced progesterone synthesis and reduced cAMP synthesis independent of the presence or absence of oocytes. BMP-7 also increased FSH-induced estradiol production, and the response was further augmented in the presence of oocytes. In contrast, BMP-6 had no impact on estradiol synthesis regardless of the presence of oocytes. Since BMP-7 changed neither forskolin- nor cAMP-induced estradiol production, the BMP-7 action was mediated through a FSH receptor signaling mechanism that was independent of cAMP-PKA pathway. Treatment with FSH, but not cAMP, activated ERK1/2 phosphorylation in granulosa cells, which was further accelerated by oocytes. A specific ERK inhibitor U0126 increased estradiol production and decreased FSH- and forskolin-induced progesterone production and cAMP synthesis. This suggests that ERK activation is directly linked to inhibition of estradiol synthesis and amplification of cAMP. Moreover, FSH-induced ERK1/2 phosphorylation was inhibited by BMP-7 but not influenced by BMP-6. On the other hand, BMP signaling including Smad1/5/8 phosphorylation and Id-1 transcription was upregulated by FSH and oocytes in granulosa cells through inhibition of Smad6/7 expression. Collectively, oocytes enhance FSH-induced MAPK activation and BMP signaling in granulosa cells, which leads to differential regulation of steroidogenesis elicited by BMPs in the presence of FSH in developing follicles.
Expression regulated by
Comment
Bone morphogenetic protein 7 (BMP-7) increases the expression of follicle-stimulating hormone (FSH) receptor in human granulosa cells. Shi J et al. OBJECTIVE: To examine the effect of bone morphogenetic protein 7 (BMP-7) on FSH receptor (FSHR) expression in human granulosa cells. DESIGN: Laboratory study using human samples. SETTING: University hospital. PATIENT(S): Human granulosa cells were obtained from 60 women undergoing oocyte retrieval for IVF. INTERVENTION(S): Human granulosa cells (GCs) were cultured with recombinant BMP-7, followed by RNA extraction. MAIN OUTCOME MEASURE(S): mRNA levels of GCs were measured by real-time reverse-transcription polymerase chain reaction. RESULT(S): Bone morphogenetic protein 7 increased FSHR gene expression in human luteinized granulosa cells, whereas it decreased LH receptor gene expression. Bone morphogenetic protein 7 also increased FSH-induced cyclic adenosine monophosphate production in GCs, indicating up-regulation of the cellular response to FSH. Although BMP-7 increased gene expression of activin-betaA and -betaB in GCs, inhibition of activin function did not affect the BMP-7-induced FSHR gene expression. CONCLUSION(S): These findings provide new insight into the biologic function of BMP-7 in the human ovary and demonstrate its unique mechanism of regulating FSHR action.
Ovarian localization
Theca
Comment
Shimasaki S, et al 1999 reported that a functional bone morphogenetic protein system exists in the ovary.
In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and
II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling
rats.
To explore the paracrine function of this BMP system, they examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating
hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both
BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically,
physiological concentrations of the BMPs enhanced and attenuated the stimulatory
action of FSH on estradiol and progesterone production, respectively. Furthermore, the BMPs increased
granulosa cell sensitivity to FSH. It was hypothesized that BMPs might be the long-sought "luteinization
inhibitor" in Graafian follicles during their growth and development.
Involvement of the bone morphogenetic protein/receptor system during follicle development in the bovine ovary: Hormonal regulation of the expression of bone morphogenetic protein 7 (BMP-7) and its receptors (ActRII and ALK-2). Shimizu T et al. Bone morphogenetic proteins (BMPs) are crucial factors in follicular growth and development. Among the BMP ligands, BMP-7 which use ActRII as their type II receptor, strongly bind to ALK-2 as their type I receptor. However, whether their receptors are expressed and the regulatory mechanisms controlling their expression during the process of bovine follicle development are still unknown. The aim of the present study was to clarify the involvement of the receptor system for BMP-7 in follicular selection by examining the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the regulation of ActRII and ALK-2 mRNA expression in bovine granulosa cells (GCs). To observe mRNA expression, follicles were obtained from heifers and GCs were classified into two groups: pre-selection follicles (PRF; follicles with an average diameter of 7mm and low E2) and post-selection follicles (POF; follicles with an average diameter of 15mm and high E2). The theca cell (TC) layer and GCs were harvested from aspirated follicles. For in vitro studies, GCs were obtained from bovine follicles of 4-7mm diameter and cultured in Dulbecco's modified Eagle's/F12 (DMEM/F-12) medium with 10% fetal calf serum for 24h. The medium was then replaced with serum-free DMEM/F-12 supplemented with different doses of E2 (1, 10,100ng/ml), FSH (1, 5, 10ng/ml) or combinations of 1ng/ml of E2 with different FSH doses (1, 5, 10ng/ml). Total RNA was extracted from GCs and the mRNA expression of ActRII and ALK-2 was estimated by the quantitative PCR method using LightCycler. The expression of BMP-7 mRNA in TCs did not differ between the PRF and POF. ActRII and ALK-2 expression was detected in GCs from bovine antral follicles and was higher in the GCs of POF than in those of PRF, while the expression of the ActRII and ALK-2 genes in the TCs was not different between PRF and POF. Treatment of GCs with E2 (10ng/ml) alone increased the expression of both ActRII and ALK-2 mRNAs, whereas FSH alone had no effect. However, ActRII and ALK-2 mRNA levels were up-regulated by the combination of E2 (1ng/ml) and FSH (5ng/ml). The results of the present study provide the first evidence that FSH and E2 regulate the expression of the ActRII and ALK-2 genes in bovine GCs. Thus, our data suggest that the BMP7/ActRII/ALK-2 system may be critically involved in the process of selection of bovine follicles.
Follicle stages
Antral, Preovulatory
Comment
BMPs and BMPRs in chicken ovary and the effects of BMP-4 and -7 on granulosa cell proliferation and progesterone production in vitro.
Onagbesan OM, et al .
This study determined the expression of the mRNA encoding for BMPs and their receptors in the chicken ovary and explored possible roles for them. The expression of the mRNA for BMP-2, -4, -6, -7 and BMPR-IA, IB, and II was determined and quantified by a semi-quantitative RT-PCR. The mRNAs for all the BMPs and receptors determined were present in both the granulosa (G) and theca (T) cells of the F1, F2 and F3 follicles. All BMP mRNAs increased in granulosa cells with follicular development whereas only BMP-7 mRNA has this trend in the theca. BMP-2, -4 and -6 mRNAs in theca were similar between follicles. BMPR-IA mRNA was similar in F2G and F3G but lower in F1G. BMPR-IB mRNA was similar in granulosa of all follicles and BMPR-II mRNA increased with development. In the theca, each receptor subtype showed equal distribution between follicles. mRNA levels for BMPR-IB and -II were higher in granulosa than in theca suggesting that the granulosa is a major target for BMPs. BMP-4 and -7 stimulated basal, IGF-I-, and gonadotrophin-stimulated progesterone production by cultured granulosa cells with differential responses between cells from the F1 and F3/4. This suggests involvement in follicular differentiation. BMP-4 and -7 reversed the inhibitory effects of TGFalpha on basal and gonadotropin-stimulated granulosa cell progesterone production with greater effect in the F1 compared to the F3/4. This effect suggests an important role for BMPs interacting with TGFalpha in modulating the effects of gonadotrophins and IGF-I on follicular differentiation. Finally, BMP-7 stimulated granulosa cell proliferation but BMP-4 inhibited TGFalpha + IGF-I or + FSH- stimulated granulosa cell proliferation suggesting a role in the control of follicular growth during development. These effects of BMP-4 and -7 on the granulosa cell function showed relationships with the expression levels of the BMPs and the BMPR-II.
Phenotypes
Mutations
2 mutations
Species: porcine
Mutation name: type: naturally occurring fertility: fertile Comment: BMP7 is a candidate gene for reproductive traits in Yorkshire sows. Du X et al. (2020) Bone morphogenetic protein 7 (BMP7) is of the BMP subfamily, and has effects on female fertility by regulating steroidogenesis, granulosa cell states, and follicular development. In the present study, there was assessment of the combined genotypes formed by the three variants within the 3'-UTR of BMP7 gene as associations with sow reproductive functions. The 3'-UTR of the BMP7 gene of pigs was identified using the 3' RACE assay, and its full-length sequence was found to be 1538 bp in length. Multiple RNA regulatory elements were detected in this region, luciferase activity assays were performed and results indicated miR-22-3p affects BMP7 by directly binding to the miRNA response element in the 3'-UTR (c.2358-2382). In addition, two novel complete linkage variants, c.2256 G > C and a 7-bp indel (c.2259-2265), were identified within the 3'-UTR of the BMP7 gene of pigs. Importantly, combined genotypes with these two novel variants and c.1569A > G, a variant previously identified in the BMP7 3'-UTR of pigs, were associated with sow reproductive traits, including the total number of piglets born, number of dead piglets at birth, and litter weight in the Yorkshire pig population studies. Results from the present study confirm that BMP7 is a candidate gene for the reproductive traits in Yorkshire sows.//////////////////
Species: mouse
Mutation name: BMP-7 null
type: null mutation fertility: embryonic lethal Comment: The Bmp7 gene was inactivated by insertional mutagenesis due to transgene integration. The Bmp7 homozygous null condition in mice is a postnatal lethal mutation and is associated with various developmental defects: holes in the basisphenoid bone and the xyphoid cartilage, retarded ossification of bones, fused ribs and vertebrae, underdeveloped neural arches of the lumbar and sacral vertebrae, polydactyly of the hind limbs, a kinked tail, a reduced number of nephrons, polycystic kidney, lack of retinal pigmentation, and retarded lens development Jena et al. 1997 .