Reproductive Genomics Program: Mutant Models for Infertility

Reproductive Genomics

From 2002 to 2012, the NIH funded the Reproductive Genomics Program at the Jackson Laboratory for Mammalian Genetics (Bar Harbor), run by Drs. John J. Eppig, Mary Ann Handel, and John Schimenti. The overall goals of this program were to produce and distribute mutant models for infertility and to investigate underlying causes in order to identify new genes and pathways contributing to reproductive function.

Production of Mutant Models for Infertility

ENU-mutagenesis of male mice was followed by a standard three-generation breeding scheme, with fertility testing of all G3 offspring in order to identify putative reproductive mutants.

Figure 1. ENU Mutagenesis Breeding Scheme

In all, almost 500 pedigrees were established; approximately 20,000 individual G3 mice were screened by phenotype analyses, and close to 50 new mutations were identified. Phenotype screening protocols covered basic aspects of reproductive histology and physiology.

Figure 2. Female Fertilty Screening

Figure 3. Male Fertility Screening

Of the mutant lines established, about 75% exhibited a male infertility phenotype only, almost 10% exhibited female infertility only, and about 15% exhibited infertility of both sexes. All mutations were mapped to a chromosome region and all were archived as cryopreserved sperm from heterozygous males. Of these, the following mutations listed in Table 1 were fine mapped and the affected genes identified.

Table 1. Reproductive Genomics Gene Mutations

Mutant Line Affected Gene
repro1 Iqcg
repro4 Mtap2
repro5 Brwd1
repro7 Prdm9
repro8 Eif4g3
repro9 Mybl1
repro11 Smc1b
repro12 Akap9
repro22 Mad2l2 (Rev7)
repro23 Tdrd12
repro27 Golga3
repro32 Capza3
repro34 Stx2
repro42 Spata22
repro57 Rnf212
ferf1 Dnahc1
Marf1 4921513D23Rik
omd1 Padi6
spcar3 Setx
vac* Chp1
*The vac line exhibited a neurological phenotype,
but not infertility

The remaining mutations were regionally mapped, as detailed in Table 2.

Table 2. Remaining Mutations

Line ID Chromosome Chromosome Region
(Ensembl Build 33)
Male Phenotype Female Phenotype
Flanking Markers Base Pair Region
repro14 Chr 5 D5Mit338 to D5Mit25 107690857-112994570

In vitro clinic: Very low testis weight, no epididymal sperm recovered.

Histology evaluation: Arrest of spermatogenesis predominantly at pachytene stage of meiotic prophase or meiotic division stage (see 1), some germ cells appear apoptotic (see 2).

In vitro clinic: No oocytes recovered.

Histology evaluation: Tiny ovaries with no growing oocytes or follicles (see 3).

repro3 Chr 10 D10Mit38 to D10Mit194 44256482-47007195

In vitro clinic: Low epididymal sperm concentration, many sperm have abnormally shaped heads, sperm tails are short, no sperm motility, no IVF success.

Histology evaluation: Abnormal spermiogenesis (see 1) characterized by abnormally shaped sperm heads and abnormal tail morphology.

Publication: Lessard C, Lothrop H, Schimenti JC, Handel MA. Mutagenesis-generated mouse models of human infertility with abnormal sperm. Hum Reprod. 2007 Jan;22(1):159-66. Epub 2006 Aug 18.

repro2 Chr 5 D5Mit68 to D5Mit65 118958939-122584115

In vitro clinic: Very low epididymal sperm concentration, many sperm have abnormally shaped heads or no heads, sperm tails are short or curled, no sperm motility, no IVF success.

Histology evaluation: Abnormal spermiogenesis (see 1) characterized by abnormally shaped spermatid heads and abnormal tail morphology.

Publication: Lessard C, Lothrop H, Schimenti JC, Handel MA. Mutagenesis-generated mouse models of human infertility with abnormal sperm. Hum Reprod. 2007 Jan;22(1):159-66. Epub 2006 Aug 18.

repro46 Chr 9 D9Mit96 to D9Mit301 50826065-56699792

In vitro clinic: Very low testis weight, low seminal vesicle weight, no epididymal sperm recovered.

Histology evaluation: Arrest of spermatogenesis during late pachytene stage of meiotic prophase (see 1), some seminiferous tubules have only spermatogonia and Sertoli cells present (see 1), some germ cells appear apoptotic (see 2).

In vitro clinic: No oocytes recovered.

Histology evaluation: Tiny ovaries with some follicle activity (see 3), hemorrhagic cysts present in some ovaries (see 4).

repro36 Chr 9 D9Mit71 to D9Mit103 50248563-60611959

In vitro clinic: Very low testis weight, low seminal vesicle weight, no epididymal sperm recovered.

Histology evaluation: Arrest of spermatogenesis during pachytene stage of meiotic prophase, some tubules are greatly depleted of germ cells (see 1).

In vitro clinic: No oocytes recovered.

Histology evaluation: Tiny ovaries with no growing oocytes or follicles (see 2).

repro34 Chr 5 D5Mit138 to D5Mit372 124262463-131316975

In vitro clinic: Very low testis weight, low seminal vesicle weight, no epididymal sperm recovered.

Histology evaluation: Abnormal spermiogenesis with accumulation of meiotic division-phase spermatocytes and degenerating round spermatids present in multinucleated cell bodies (see 1).

repro33 Chr 16 D16Mit58 to D16Mit106 31121394-84904036

In vitro clinic: Low seminal vesicle weight, low epididymal sperm concentration, abnormal looped sperm tails (see 1), sperm have very low motilty, very low IVF success.

Histology evaluation: Normal spermatogenesis (see 2).

repro29 Chr 5 D5Mit240 to D5Mit98 108167726-137190943

In vitro clinic: Low seminal vesicle weight, low epididymal sperm concentration, abnormally shaped sperm heads with and without tails, very low sperm motility, very low IVF success.

Histology evaluation: Abnormal spermiogenesis (see1-3) with abnormal spermatid head shape (see 2), low number of elongating spermatids (see 3).

repro19 Chr 16 D16Mit165 to D16Mit103 12732404-30793475

In vitro clinic: Low testis weight, low seminal vesicle weight, low epididymal sperm concentration, abnormally shaped sperm heads with missing or short sperm tails, variable motility, very low IVF success.

Histology evaluation: Abnormal spermatogenesis (see 1-3) with low/abnormal spermatogonia and spermatocytes. Abnormal spermiogenesis with abnormal spermatid heads (see 2), low number of spermatids in testis lumen (see 3).

Hormone analysis: High levels of luteinizing hormone and testosterone.

repro 26 Chr 3 D3Mit221 to D3Mit179 7875675-31412338

In vitro clinic: Low testis weight, very low epididymal sperm concentration, abnormally shaped sperm heads with and without tails, very low motility, very low IVF success.

Histology evaluation: Abnormal spermiogenesis (see 1-3) with abnormal ""hammer-head"" shape of elongating spermatid heads (see2-3), low number of spermatids in testis lumen (see 2-3).

repro28 Chr 9 D9Mit299 to D9Mit259 49538217-69961900

In vitro clinic: Normal testis weight, low seminal vesicle weight, low epididymal sperm concentration, abnormally shaped sperm heads, sperm with kinked tails and without tails, very low sperm motility, very low IVF success.

Histology evaluation: Abnormal spermiogenesis (see 1-3) with abnormal spermatid head shape (see 2-3).

repro24 Chr 1 D1Mit100 to D1Mit113 143741638-171733320

In vitro clinic: Low epididymal sperm concentration, low sperm motility, abnormally shaped sperm heads, no IVF success.

Histology evaluation: Normal

repro20 Chr 9 D9Mit89 to D9Mit299 29368996-49538217

In vitro clinic: Low testis weight, very low epididymal sperm concentration, poor sperm motility, abnormally shaped sperm heads with short or absent tails, no IVF success.Histology evaluation: Abnormal spermiogenesis with abnormal elongated spermatid heads (see 1-2), loss of sperm tails (see 1).

repro17 Chr 17 D17Mit175 to D17Mit66 30343204-45241036

In vitro clinic: Low epididymal sperm concentration, low sperm motility, abnormally shaped sperm heads with short tails, poor IVF success. Histology evaluation: Primarily normal spermatogenesis. Some abnormal spermiogenesis with abnormal spermatid head shape (see 1).

repro21 Chr 17 D17Mit197 to D17Mit66 18003678-45241036

In vitro clinic: Low testes weight, very low epididymal sperm concentration, no sperm motility, abnormally shaped sperm heads with no tails, no IVF success. Histology evaluation: Abnormal spermiogenesis, with abnormal spermatid heads without tails (see 1-2), low number of spermatids in testis lumen (see 3).

repro16 Chr 8 D8Mit100 to D8Mit359 55715457-71351071

In vitro clinic: Low epididymal sperm concentration, low sperm motility, abnormal sperm head shape and large sperm, poor IVF success.

Histology evaluation: Abnormal spermiogenesis with few elongated spermatids in some tubules (see 1), abnormal heads (see 2), abnormal spermatid elongation (see 3) and abnormal cells in the epididymal lumen (see 4).

repro15 Chr 11 D11Mit231 to D11Mit140 35481820-53956337

In vitro clinic: low body weight, all epididymal sperm have abnormal head morphology, no IVF success.

Histology evaluation: Abnormal spermiogenesis with abnormal shaped spermatid heads abundant in testis (see 1), few elongated spermatids in some tubules (see 2), severe spermatogenesis impairment in some tubules (see 3); abnormal cells in the epididymal lumen (see 4).

repro10 Chr 4 D4Mit164 to D4Mit178 59345690-66273219

In vitro clinic: Low body weight, variable epididymal sperm concentration, low sperm motility, low IVF success.

Histology evaluation: Spermatogenesis predominantly normal with some abnormal spermiogenesis reflected in spermatid heads.

repro13 Chr 7 D7Mit76 to D7Mit247 15863631-32307081

In vitro clinic: Low epididymal sperm concentration, abnormal sperm heads, low IVF success.

Histology evaluation: Abnormal spermiogenesis with elongated spermatid heads (see 1).

All mutant lines are publicly available by request. Lines have been distributed to investigators in the U.S., Europe, Japan and China.

Publications Reporting Reproductive Genomics Mutants

Ward, J, L Reinholdt, S Hartford, L Wilson, R Munroe, K Schimenti, B Libby, M O’Brien, J Pendola, JJ Eppig, J Schimenti. 2003. Towards the genetics of mammalian reproduction: induction and mapping of gametogenesis mutants in mice. Biol Reprod 69:1615-25.

Lessard C, JK Pendola, SA Hartford, JC Schimenti, MA Handel, JJ Eppig.  2004. New mouse genetic models for human contraceptive development.  Cytogenet Genome Res 105:222-227.

Handel, MA, C Lessard, L Reinholdt, J Schimenti, JJ Eppig.  2006. Mutagenesis as an unbiased approach to identify novel contraceptive targets.  Mol Cell Endo 250:201-205.

Bannister L, R Pezza, J Donaldson, D de Rooij, K Schimenti, D Camerini-Otero, JC Schimenti. 2007. Male-specific sterility in mice carrying a dominant, recombination-defective allele of the RecA homolog Dmc1. PLoS Biol 5:e105. doi:10.1371/journal.pbio.0050105. PMID: 17425408.

Furnes B, JC Schimenti. 2007. Fast forward to new genes in mammalian reproduction. J Physiol 578:25-32. doi: 10.1113/jphysiol.2006.119164. PMID: 16973708.

Lessard C, H Lothrop, JC Schimenti, MA Handel.  2007.  Mutagenesis-generated mouse models of human infertility with abnormal sperm.  Hum Reprod 22:159-166.

Akiyama K, S Akiyama, Y Asano, M Khalaj, C Kiyosu, AA Masoudi, S Takahashi, K Katayama, T Tsuji, J Noguchi, T Kunieda. 2008. A new ENU-induced mutant mouse with defective spermatogenesis caused by a nonsense mutation of the Syntaxin 2/Epimorphin (Stx2/Epim) gene. J Reprod Dev 54:122-128.

Khalaj M, AE Abassi, R Nishimuta, K Akiyama, T Tsuji, J Noguchi, T Kunieda. 2008. Leydig cell hyperplasia is an ENU-induced mutant mouse with germ-cell depletion J Reprod Dev 54:225-228.

Philipps, D., K Wigglesworth, S Hartford, F Sun, S Pattabiraman,  K Schimenti, MA Handel,  JJ Eppig, J Schimenti.  2008.  The dual bromodomain and WD repeat-containing mouse protein BRWD1 is required for normal spermiogenesis and the oocyte-embryo transition. Devel. Biol. 317:72-82.

Asano Y, K Akiyama, T Tsuji, S takahashi, J Noguchi, T Kunieda. 2009. Characteriztion and linkage mapping of an ENU-induced mutant mouse with defective spermatogenesis. Exp. Anim. 58:525-532.

Geyer CB, AL Inselman, JA Sunman, S Bornstein, MA Handel, EM Eddy. 2009. A missense mutation in the Capza3 gene and disruption of F-actin organization in spermatids of repro32 infertile male mice. Dev Biol 330:142-152.

Handel MA, JC Schimenti. 2010. Genetics of mammalian meiosis: regulation, dynamics and impact on fertility. Nat Rev Genet 11:124-136.

Li S, A Francisco, C Han, S Pattabiraman, M Foote, S Giesy, C Wang, J Schimenti, Y Boisclair, Q Long.  2010. The full-length Isoform of pleckstrin homology domain-interacting protein (PHIP) is required for postnatal growth. FEBS Letters, 584:4121-4127. PMCID: PMC2965186

Sun F, K Palmer, MA Handel. 2010. Mutation of Eif4g3, encoding a eukaryotic translation initiation factor, causes male infertility and meiotic arrest of mouse spermatocytes. Development 137:1699-1707.

Bolcun-Filas, E., L Bannister, A Barash, K, S Hartford, JJ Eppig, MA Handel, L Shen, J Schimenti. 2011. A-MYB is a master regulator of meiosis in male mice.  Development 138:3319-3330.

Sun F, MA Handel. 2011. A mutation in Mtap2 is associated with arrest of mammalian spermatocytes before the first meiotic division. Genes 2(1):21-25. doi:10.3390/genes2010021

Bolcun-Filas E, J Schimenti. 2012. Genetics of meiosis and recombination in mice. Intl Rev Cell Molec Biol 298:179-227.

Guan Y, D Gorenshteyn, JC Schimenti, MA Handel, CJ Bult, MA Hibbs, OG Troyanskaya. 2012. Tissue-specific functional networks for prioritizing disease genes. PloS Comp Biol 8:e1002694.

La Salle S., K Palmer, M O'Brien, J Schimenti, JJ Eppig, MA Handel. 2012. Spata22, a novel vertebrate-specific gene, is required for meiotic progress in mouse germ cells. Biol Reprod 86:45. PMID22011390

Su, Y-Q, K Sugiura, F Sun, J Pendola, G Cox, MA Handel, J Schimenti, JJ Eppig. 2012. MARF1 regulates essential oogenic processes in mice.  Science, 335:1496-9.

Su, Y-Q, F Sun, MA Handel, JC Schimenti, JJ Eppig. 2012. Meiosis arrest female 1 (MARF1) has nuage-like function in mammalian oocytes. Proc Natl Acad Sci USA 109:18653-18660.

Bentson, LF, VA Agbor, LN Agbor, AC Lopez, LE Nfonsam, SS Bornstein, MA Handel, CC Linder.  2013. New point mutation in Golga3 causes multiple defects in spermatogenesis. Androl 1:440-450.

Fujiwara, Y, N Ogunuke, K Inouye, A Ogura, MA Handel, J Noguchi, T Kunieda. 2013. t-SNARE Syntaxin2 (STX2) is implicated in intracellular transport of sulfoglycolipids during meiotic prophase in spermatogenesis. Biol Reprod 88:141, 1-9.

Li, X, C Roy, X Dong, E Bolcun-Filas, J Wang, B Han, J Xu, M Moore, J Schimenti, Z Weng, P Zamore. 2013. An ancient transcription factor initiates the burst of piRNA production during meiosis in the mouse testis. Molec Cell 50:67-81.

Liu Y, HC Zaun, J Orlowski, SL Ackerman. 2013. CHP1-mediated NHE1 biosynthetic maturation is required for Purkinje cell axon homeostasis. J Neurosci 33:12656-12669.

Luo M, F Yang, NA Leu, J Landaiche, MA Handel, R Benavente, S La Salle, PJ Wang. 2013. MEIOB exhibits single-stranded DNA-binding and exonuclease activities and is essential for meiotic recombination.  Nat Commun 2013 Nov 18; 4:2788.

Pandey RR, Y Tokuzawa, E Hayashi T Ichisaka, S Kajita, Y Asano, T Kunieda, R Sachidanandam, S Chuma, S Yamanaka, RS Pillai. 2013. Tudor domain containing 12 (TRD12) is essential for secondary PIWI interacting RNA biogenesis in mice. Proc Natl Acad Sci 110:16492-16497.

Schimenti K, S Feuer, L Griffin, N Graham, C Bovet, S Hartford, J Pendola, C Lessard, J Schimenti, J Ward.  2013. AKAP9 is vital for spermatogenesis and Sertoli cell maturation in mice. Genetics 194:447-457.

Harris T, K Schimenti, R Munroe, J Schimenti. 2014. IQ motif-containing G (Iqcg) is required for mouse spermiogenesis. G3: Genes, Genome, Genetics 4:367-372. doi:10.1534/g3.113.009563. PMID: 24362311.

Abbasi A, M Khalaj, K Akiyama, Y Mukai, H Matsumoto, N Said, M Yoshida, T Kunieda. 2015. Lack of Rev7 function results in development of tubulostromal adenomas in mouse ovary. Mol Cell Endocrinol 412:19-25.

Fujiwara Y, H Matsumoto, K Akiyma, A Srivastava, M Chikushi, MA Handel, T Kunieda. 2015. An ENU-induced mutation in the mouse Rnf212 gene is associated with male meiotic failure and infertility. Reproduction 149:67-74.

Khalaj M, A Abbasi, H Yamanishi, K Akiyama, S Wakitani, S Kikuchi, M Hirose, M Yuzuriha, M Magari, HA Degheidy, K Abe, A Ogura, H Hashimoto, T Kunieda. 2015. A missense mutation in Rev7 disrupts formation of Polz impairing mouse development and repair of genotoxic agent-induced DNA lesions. J Biol Chem 289:3811-3824.

Pattabiraman S, C Baumann, D Guisado, JJ Eppig,  JC Schimenti, R De La Fuente. Mouse BRWD1 is critical for spermatid postmeiotic transcription and female meiotic chromosome stability. 2015. J Cell Biol. 208(1):53-69.

Sun F, Fujiwara Y, Reinholdt LG, Hu J, Saxl RL, Baker CL, Petkov PM, Paigen K, MA Handel. 2015. Nuclear localization of PRDM9 and its role in meiotic chromatin modifications and homologous synapsis. Chromosoma Apr 18;PubMed PMID: 25894966.