Spermatogonial asynchrony in Tex14 mutant mice lacking intercellular bridges.

The existence of cytoplasmic passages between germ cells and their potential function in the control of the spermatogenic process has long been an intriguing question. Evidence of the important role of such structures, known as intercellular bridges (ICB), in spermatogenesis has been implicated by the failure of spermatogenesis in testis-expressed gene 14 (Tex14) mutant mice, which lack the ICBs, to progress past the pachytene spermatocyte stage. Using these Tex14 mutants, the present study evaluated, for the first time, the behavior and synchrony of the spermatogonial lineage in the absence of ICBs. Our data suggest that the absence of these cytoplasmic connections between cells affects the expansion of the undifferentiated type A (Aundiff) spermatogonia compartment and their transition to A1, resulting in a significant numerical reduction of differentiating A1 spermatogonia, but did not interfere with cell amplification during subsequent mitotic steps of differentiating spermatogonia from A1 through intermediate (In). However, beginning at the type B spermatogonia, the synchrony of differentiation was impaired as some cells showed delayed differentiation compared to their behavior in a normal seminiferous epithelium cycle. Thus although spermatogonial development is able to proceed, in the absence of ICBs in Tex14-/- mutants, the yield of cells, specific steps of differentiation, the synchrony of the cell kinetics, and the subsequent progression in meiosis are quantitatively lower than normal.

Nine genes abundantly expressed in the epididymis are not essential for male fecundity in mice.

BACKGROUND: Spermatozoa become competent for fertilization during transit through the epididymis. As spermatozoa from the proximal caudal epididymis can fertilize eggs, proteins from the caput and corpus epididymis are required for sperm maturation. OBJECTIVES: Microarray analysis identified that more than 17,000 genes are expressed in the epididymis; however, few of these genes demonstrate expression restricted to the epididymis. To analyze epididymis-enriched gene function in vivo, we generated knockout (KO) mutations in nine genes that are abundantly expressed in the caput and corpus region of the epididymis. MATERIALS AND METHODS: KO mice were generated using the CRISPR/Cas9 system. The histology of the epididymis was observed with hematoxylin and eosin staining. KO males were caged with wild-type females for 3-6 months to check fertility. RESULTS: We generated individual mutant mouse lines having indel mutations in Pate1, Pate2, or Pate3. We also deleted the coding regions of Clpsl2, Epp13, and Rnase13, independently. Finally, the 150 kb region encoding Gm1110, Glb1l2, and Glb1l3 was deleted to generate a triple KO mouse line. Histology of the epididymis and sperm morphology of all KO lines were comparable to control males. The females mated with these KO males delivered pups at comparable numbers as control males. DISCUSSION AND CONCLUSION: We revealed that nine genes abundantly expressed in the caput and corpus epididymis are dispensable for sperm function and male fecundity. CRISPR/Cas9-mediated KO mice generation accelerates the screening of epididymis-enriched genes for potential functions in reproduction.

Contemporary genetic technologies and female reproduction.

BACKGROUND: The Fifth Evian Annual Reproduction (EVAR) Workshop Meeting discussed knowledge regarding contemporary genetics in female reproduction. METHODS: Specialist reproductive medicine clinicians and geneticists delivered presentations based on published literature and current research. The content of this report is based on the expert presentations and subsequent group discussions that took place during this Workshop. RESULTS: Numerous ovarian genes with a role in infertility have been identified. Future challenges for genetic screening of patients, such as those with polycystic ovary syndrome, primary ovarian insufficiency or endometriosis, include the identification of high-throughput strategies and how to apply these findings to infertile patients. The identification of high-quality embryos in IVF using objective technologies remains a high priority in order to facilitate single-embryo transfer. Gene expression profiling of cumulus cells surrounding the oocyte, and proteomic and metabolomic approaches in embryo culture media may significantly improve non-invasive embryo quality assessment. CONCLUSIONS: The way forward in advancing the knowledge of genes involved in reproduction was considered to be through genome-wide association studies involving large numbers of patients. Establishing international collaboration is required to enable the application of such technologies in sufficient numbers of patients.

Oocyte-somatic cell communication and microRNA function in the ovary.

An enormous amount of knowledge about the ovary has been generated over the last two decades, due in part to the development of strategies to genetically manipulate the mouse using embryonic stem cell technology. Our group and others have identified multiple factors that are important and essential at all stages of ovarian folliculogenesis from formation of the primordial factor to ovulation. It is obvious that an oocyte, the key cargo of the ovary, and the surrounding granulosa cells, the support cells of the follicle, entertain a dialog that is key for granulosa growth and differentiation and oocyte growth, maturation, and fertilization. In addition to the involvement of genes in these processes, small non-coding RNAs including microRNAs and siRNAs have been implicated as key regulators, especially in the oocyte. These studies have direct implications for human fertility control in the assisted reproductive technology (ART) laboratory.

The power of mouse genetics to study spermatogenesis.

Approximately 80 million people worldwide are infertile, and nearly half of all infertility cases are attributed to a male factor. Therefore, progress in reproductive genetics becomes crucial for future diagnosis and treatment of infertility. In recent years, enormous progress has been made in this field. More than 400 mutant mouse models with specific reproductive abnormalities have been produced, and numerous human association studies have been discovered. However, the translation of basic science findings to clinical practice remains protracted, with only modest progress in the application of novel findings to clinical genetic testing and cures. To date, the most significant findings in male infertility remain numeric and structural chromosomal abnormalities and Y-chromosome microdeletions in infertile men. Thus, we anticipate that future genetic investigations will focus on infertile men with a normal somatic karyotype but with various spermatozoal defects, like insufficient production of spermatozoa (oligozoospermia), inadequate motility (asthenozoospermia), abnormal morphology (teratozoospermia), or combinations of these defects. Ultimately, basic advances in mammalian nonhuman reproduction will translate to clinical advances in human reproduction and testing for infertile humans, thereby helping to improve diagnostics and health care for infertile patients.

Spermatogenesis does not require the local production of follistatin.

It has been proposed that follistatin can modulate the actions of activins and/or other members of the transforming growth factor-beta superfamily of proteins on testicular function, since mice overexpressing follistatin showed spermatogenic disruption. However, since mice with targeted disruption of the follistatin gene die soon after birth, it is not feasible to determine the effect of the absence of follistatin on testicular function using this model. To further understand the role of follistatin on the development and maintenance of spermatogenesis, fetal testes, collected by Caesarean section at day 18 of gestation from follistatin null mice, were transplanted to the external ear of castrated recombination activating gene 1 immunocompromised male mice. The testicular grafts were then analysed 7-8 weeks after transplantation and showed that full spermatogenesis developed in both the testes of wild-type and follistatin null mice. This study indicates that, if follistatin is required to modulate spermatogenic development, it is not supplied by local testicular production but by circulating follistatin from the host mouse.

Sohlh1 is essential for spermatogonial differentiation.

Spermatogonia are adult germline stem cells that can both self-renew and differentiate into spermatocytes. Little is known about factors necessary for spermatogonial differentiation. We identified a novel germ-cell-specific transcription factor that we named Sohlh1 (testis and ovary expressed basic helix-loop-helix (bHLH) transcription factor). In males, Sohlh1 is preferentially expressed in prespermatogonia and Type A spermatogonia. Loss of Sohlh1 causes infertility by disrupting spermatogonial differentiation into spermatocytes. Seven-day-old testes without Sohlh1 still express the testis-specific transcription factors Etv5, Taf4b, Zfp148, and Plzf, overexpress a novel Tohlh2 bHLH transcription factor, but lack LIM homeobox gene Lhx8 and show reduced expression of Ngn3. Sohlh1 represents the first testis-specific bHLH transcription factor that is essential for spermatogonial differentiation.

Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF.

BACKGROUND: A biochemical marker for embryo development would increase the chance of a successful pregnancy with IVF by optimizing oocyte and embryo selection, and allow fewer embryos to be transferred. In this study, we correlated cumulus granulosa cell gene expression of hyaluronic acid synthase 2 (HAS2), cyclooxygenase 2 (COX2; PTGS2) and gremlin (GREM1) with subsequent embryo development in search of a parameter for embryo selection. METHODS: Cumulus cell gene expression was determined prospectively on eight consecutive patients undergoing IVF with ICSI. Immediately following oocyte retrieval, the cumulus was stripped from the oocyte, and cumulus gene expression for PTGS2, HAS2 and GREM1 was assessed using a one-step real-time quantitative RT-PCR assay. Oocyte quality, fertilization and embryo morphology were correlated to relative gene expression. RESULTS: Gene expression data were available on cumulus cells from 108 oocytes that developed into 70 embryos (64.8% fertilization rate). Cumulus PTGS2, HAS2 and GREM1 expression was higher from oocytes that developed into higher quality embryos (grades 3, 4 and 5) compared with lower quality embryos (grades 1 and 2) (P<0.05, P<0.001 and P<0.001, respectively). HAS2 and GREM1 expression was also higher from the cumulus surrounding oocytes that gave rise to higher grade embryos (P<0.001). The expression of PTGS2 and HAS2 was 6-fold higher, and that of GREM1 was 15-fold higher in cumulus yielding higher grade embryos versus lower grade embryos. CONCLUSION: PTGS2, HAS2 and GREM1 gene expression correlates to morphological and physiological characteristics and provides a novel approach to predict human embryo development. Ultimately, with better predictors of follicular and embryonic health, higher quality embryos can be selected and transferred, reducing higher order pregnancy rates.

Oosp1 encodes a novel mouse oocyte-secreted protein.

Oocyte-somatic cell communication is necessary for normal ovarian function. However, the identities of the majority of oocyte-secreted proteins remain unknown. A novel cDNA encoding mouse oocyte-secreted protein 1 (OOSP1) was identified using a modified subtractive hybridization screen. The Oosp1 cDNA encodes a 202-amino acid protein that contains a 21-amino acid signal peptide sequence, 5 putative N-linked glycosylation consensus sequences, and 6 cysteines that are predicted to form 3 disulfide bonds. OOSP1 shares amino acid identity with placental-specific protein 1 (PLAC1), a secreted protein expressed in the placenta and the ectoplacental cone. The Oosp1 mRNA is approximately 1.0 kb and is present at high levels in the oocytes of adult ovaries and at lower levels in the spleen. The mouse Oosp1 gene is 5 exons, spans greater than 16.4 kb, and localizes to chromosome 19 at a position that shares synteny with human chromosome 11q12-11q13. The identification of OOSP1 as a new oocyte-secreted protein permits future in vitro and in vivo functional analyses to define its role in ovarian folliculogenesis.

Anti-Müllerian hormone attenuates the effects of FSH on follicle development in the mouse ovary.

Although ovarian follicle growth is under the influence of many growth factors and hormones of which FSH remains one of the most prominent regulators. Therefore, factors affecting the sensitivity of ovarian follicles to FSH are also important for follicle growth. The aim of the present study was to investigate whether anti-Müllerian hormone (AMH) has an inhibitory effect on follicle growth by decreasing the sensitivity of ovarian follicles to FSH. Furthermore, the combined action of AMH and FSH on ovarian follicle development was examined. Three different experiments were performed. Using an in vitro follicle culture system it was shown that FSH-stimulated preantral follicle growth is attenuated in the presence of AMH. This observation was confirmed by an in vivo experiment showing that in immature AMH-deficient females, more follicles start to grow under the influence of exogenous FSH than in their wild-type littermates. In a third experiment, examination of the follicle population of 4-month-old wild-type, FSH beta-, AMH-, and AMH-/FSH beta-deficient females revealed that loss of FSH expression has no impact on the number of primordial and preantral follicles, but the loss of inhibitory action of AMH on the recruitment of primordial follicles in AMH-deficient mice is increased in the absence of FSH. In conclusion, these studies show that AMH inhibits FSH-stimulated follicle growth in the mouse, suggesting that AMH is one of the factors determining the sensitivity of ovarian follicles for FSH and that AMH is a dominant regulator of early follicle growth.

Discovery of germ cell-specific transcripts by expressed sequence tag database analysis.

OBJECTIVE: To identify transcripts whose expression is restricted to germ cells. DESIGN: Expressed sequence tags (ESTs) from unfertilized egg libraries were utilized to perform in silico subtraction and identify germ cell-specific transcripts. SETTING: Baylor College of Medicine, Houston, Texas. ANIMAL(S): C57BL/6J/129SvEv hybrid. INTERVENTION(S): Tissue harvesting from mice. MAIN OUTCOME MEASURE(S): Identification of germ cell-specific transcripts. RESULT(S): We have used the Unigene collection of mouse cDNA libraries to identify ESTs derived from unfertilized egg libraries. A total of 3,499 ESTs were identified from Knowles Solter and Ko unfertilized egg cDNA libraries. In silico subtraction identified 258 ESTs, which were found in these unfertilized egg libraries, but not in adult mouse tissue cDNA libraries. We performed reverse transcription polymerase chain reaction (RT-PCR) on multiple adult tissues with 43 selected ESTs and found 5 of them where expression was absent in heart, lung, liver, brain, spleen, stomach, intestines, kidneys, and uterus, but restricted to ovaries and testes. Three ESTs were further analyzed, and they were exclusively localized to the oocytes by in situ hybridization. CONCLUSION: We have shown that utilization of publicly available ESTs from murine EST libraries is a simple and rapid in silico approach to the identification of transcripts preferentially expressed in germ cells.

Blockage of the rete testis and efferent ductules by ectopic Sertoli and Leydig cells causes infertility in Dax1-deficient male mice.

DAX-1, an X-linked member of the orphan nuclear receptor superfamily of transcription factors, plays a key role in sex determination and gonadal differentiation. Dax1-deficient male mice are infertile and have small testes despite normal serum levels of T and gonadotropins. Examination of Dax1-deficient testes reveals dilated seminiferous tubules and abnormal parameters of sperm fertilizing capability consistent with a possible obstruction in the testis. To test this hypothesis, we performed a comprehensive evaluation of the male reproductive tract in Dax1-deficient mice. Light and electron microscopic examination revealed the rete testis is blocked by aberrantly located Sertoli cells, creating a tailback of necrosing sperm in the testis. Sertoli cells also obstruct the proximal and middle efferent ductules, and this is accompanied by an overgrowth of the efferent duct epithelium. Seminiferous tubules close to the rete testis contain ectopic Leydig cells, distinct from the hyperplastic Leydig cells present in the interstitial space. The peritubular tissue surrounding these tubules is frequently abnormal, containing relatively undifferentiated myoid cells and no basement membrane between the myoid cells and Sertoli cells. A third of aged (>1-yr-old) Dax1-deficient male mice develop sex cord-stromal tumors, derived from cells of the Sertoli/granulosa cell or Leydig cell lineages. Combined, these observations reveal abnormal differentiation and proliferation of Leydig cells and Sertoli cells in Dax1-deficient male mice, leading to obstruction of the rete testis and infertility.

Eggs in the balance.

Polycyclic aromatic hydrocarbons (PAH), found in cigarette smoke and air pollution, interact with the aryl hydrocarbon receptor (Ahr) to cause reproductive defects. Mice lacking either Ahr or the pro-apoptotic protein Bax have an increased number of primordial follicles, and these mutant oocytes are resistant to PAH toxicity. A new study shows that the Bax promoter contains two core Ahr response elements, which are required for PAH stimulation of Bax promoter activity in oocytes. Thus, the toxic effects of PAH in oocytes are mediated directly by Ahr induction of the Bax pathway.

Disruption of gamma-glutamyl leukotrienase results in disruption of leukotriene D(4) synthesis in vivo and attenuation of the acute inflammatory response.

To study the function of gamma-glutamyl leukotrienase (GGL), a newly identified member of the gamma-glutamyl transpeptidase (GGT) family, we generated null mutations in GGL (GGL(tm1)) and in both GGL and GGT (GGL(tm1)-GGT(tm1)) by a serial targeting strategy using embryonic stem cells. Mice homozygous for GGL(tm1) show no obvious phenotypic changes. Mice deficient in both GGT and GGL have a phenotype similar to the GGT-deficient mice, but approximately 70% of these mice die before 4 weeks of age, at least 2 months earlier than mice deficient only in GGT. These double-mutant mice are unable to cleave leukotriene C(4) (LTC(4)) to LTD(4), indicating that this conversion is completely dependent on the two enzymes, and in some organs (spleen and uterus) deletion of GGL alone abolished more than 90% of this activity. In an experimental model of peritonitis, GGL alone is responsible for the generation of peritoneal LTD(4). Further, during the development of peritonitis, GGL-deficient mice show an attenuation in neutrophil recruitment but not of plasma protein influx. These findings demonstrate an important role for GGL in the inflammatory response and suggest that LTC(4) and LTD(4) have distinctly different functions in the inflammatory process.

Studying TGF-beta superfamily signaling by knockouts and knockins.

The transforming growth factor beta (TGF-beta) superfamily has profound effects on many aspects of animal development. In the last decade, our laboratory and others have performed in vivo functional studies on multiple components of the TGF-beta superfamily signal transduction pathway, including upstream ligands, transmembrane receptors, receptor-associated proteins and downstream Smad proteins. We have taken gene knockout approaches to generate null alleles of the genes of interest, as well as a gene knockin approach to replace the mature region of one TGF-beta superfamily ligand with another. We found that activin betaB, expressed in the spatiotemporal pattern of activin betaA, can function as a hypomorphic allele of activin betaA and rescue the craniofacial defects and neonatal lethal phenotype of activin betaA-deficient mice. With the knockout approach, we have shown that the expression pattern of a component in the TGF-beta superfamily signal transduction cascade does not necessarily predict its in vivo function. Two liver-specific activins, activin betaC and activin betaE are dispensable for liver development, regeneration and function, whereas ubiquitously expressed Smad5 has specific roles in the development of multiple embryonic and extraembryonic tissues.

Male reproductive phenotypes in double mutant mice lacking both FSHbeta and activin receptor IIA.

Activins are known to signal through two serine/threonine kinase type II receptors. Activin receptor IIA is widely expressed in the male reproductive axis, including the pituitary and testis. Our previous studies using gene knockout mice have confirmed the essential in vivo role of activin receptor IIA in FSH homeostasis. Activin receptor IIA-null male mice are fertile, have suppressed pituitary and serum FSH levels, and demonstrate a decrease in testis size as a result of reduced Sertoli cells and germ cells. Similarly, FSHbeta null male mice are fertile despite reduced testis size and Sertoli cell number. To define the direct roles of activin receptor IIA signaling locally in the testis, independent of its effects on FSH homeostasis, we generated double mutant mice lacking both activin receptor IIA and FSH by a genetic intercross and analyzed the male reproductive phenotypes. The double mutant male mice lacking both FSH and activin receptor IIA are fertile, demonstrate no significant reduction in testis size, and produce small litters compared with mice lacking either FSH or activin receptor IIA alone. Histological analyses of the testes from double mutant mice revealed the presence of normal stages of spermatogenesis. However, there was a significant reduction in the epididymal sperm number compared with that of the individual mutants. Northern blot analyses of total RNA from testes of double mutants did not reveal transcriptional up-regulation of activin receptor IIB, the other activin type II receptor. Although RNA expression profiles of many testis cell-specific markers are unaltered, stereological analysis of the testes from double mutants indicates that there was a reduction in type A and I spermatogonial number compared with that observed in individual mutants. Our results provide in vivo genetic evidence to demonstrate that activin receptor IIA signaling plays an important local role within the testis, independent of its actions via FSH homeostasis in the pituitary.

Analysis of ovarian gene expression in follicle-stimulating hormone beta knockout mice.

FSH is a heterodimeric glycoprotein hormone that is produced in the gonadotroph cells of the anterior pituitary. It acts on Sertoli cells of the testis and granulosa cells of the ovary. We previously demonstrated that FSHbeta knockout female mice are infertile due to a block in folliculogenesis preceding antral stage development. To investigate aberrations of ovarian gene regulation in the absence of FSH, we analyzed the expression of several important marker genes using Northern blot and in situ hybridization techniques. Key findings are as follows: 1) Follicles of FSHbeta knockout mice develop a well organized thecal layer, which is positive for P450 17alpha-hydroxylase and LH receptor messenger RNAs (mRNAs). This indicates that theca recruitment is completed autonomously with respect to FSH. 2) Granulosa cells in FSH-deficient mice demonstrate an increase in FSH receptor mRNA, and decreases in P450 aromatase, serum/glucocorticoid-induced kinase, and inhibin/activin subunit mRNAs. These data support studies that implicate FSH signaling cascades in the expression of these genes. 3) In contrast to the thecal layer, granulosa cell populations in FSHbeta knockout mice do not accumulate LH receptor mRNA. This suggests that although the granulosa cells have a block in proliferation at the antral follicle stage in the absence of FSH, they do not initiate programs of terminal differentiation as seen in luteinizing cells of wild-type ovaries. 4) Ovaries of FSH-deficient mice demonstrate a modest decrease in cyclin D2 mRNA, without up-regulation of cell cycle inhibitor mRNAs associated with luteinization (i.e. p15, p27, and p21). Although components of the FSH null phenotype may be caused by partial cyclin D2 loss of function, these findings indicate that the mechanisms of granulosa cell cycle arrest in FSHbeta knockout mice are distinct from those of cycle withdrawal at luteinization. Underscoring the usefulness of the FSH-deficient mouse model, this study clarifies aspects of gonadotropin-dependent folliculogenesis, thecal layer development, cycle control in granulosa cells, and luteinization.

Analysis of the testicular phenotype of the follicle-stimulating hormone beta-subunit knockout and the activin type II receptor knockout mice by stereological analysis.

This study evaluated the role of FSH and activin A on testicular function using quantitative stereological analysis of testicular cell types in mice with targeted disruption of genes encoding the FSH beta-subunit and the activin type IIA receptor (ActRIIA). Using the optical dissector technique, the numbers of Sertoli cells and germ cells per testis were determined. Testis weights in homozygous males lacking the FSHbeta gene or the ActRIIA gene were decreased approximately 60% compared with wild-type or respective heterozygotes. Sertoli cell numbers decreased in both homozygous mice by 30-39%, and there was a comparable decline in germ cell numbers in both models. The degree of germ cell attrition increased in the later stages of spermatogenesis from a 46% reduction of spermatogonia to a 60% decrease in round spermatids. As the FSH levels are decreased in both models, the cellular lesion in both is most likely due to the FSH deficiency. Although the decrease in the Sertoli cell complement represents one cause of lower germ cell numbers, the ability of Sertoli cells to nurture germ cells is compromised by the lower FSH levels, as shown by a decrease in the round spermatid to Sertoli cell ratios in both homozygous models. We conclude that the defects in FSH beta-subunit gene knockout and ActRIIA knockout mice are related to diminished FSH action on both Sertoli cell proliferation and the capacity of Sertoli cells to nurture germ cells.

Smad5 is required for mouse primordial germ cell development.

Smad5, together with Smad1 and Smad8, have been implicated as downstream signal mediators for several bone morphogenetic proteins (BMPs). Recent studies have shown that primordial germ cells (PGCs) are absent or greatly reduced in Bmp4 or Bmp8b mutant mice. To define the role of Smad5 in PGC development, we examined PGC number in Smad5 mutant mice by Oct4 whole-mount in situ hybridization and alkaline phosphatase staining. We found ectopic PGC-like cells in the amnion of some Smad5 mutant mice, however, the total number of PGCs was greatly reduced or completely absent in Smad5 mutant embryos, similar to Bmp4 or Bmp8b mutant embryos. Therefore, Smad5 is an important factor involved in PGC generation and localization.